PreAnalytiX Blood miRNA Kit, PAXgene Blood RNA Tubes Handbook

May 2009

PAXgene

®

B l o o d m i R N A K i t H a n d b o o k

For manual or automated purification of miRNA from whole blood

Important: To be used in conjunction with

PAXgene Blood RNA Tubes.

For Research Use Only. Not for use in diagnostic procedures.

Trademarks: PAXgene

®

, PreAnalytiX

®

(PreAnalytiX GmbH); QIAGEN

®

, QIAcube

®

, QIAxcel

®

, Quantiscript

®

, QuantiTect

®

(QIAGEN Group); BD Hemogard ™ ,

BD Vacutainer

®

, Safety-Lok ™ (Becton Dickinson and Company, Franklin Lakes, NJ, USA); Affymetrix

®

, GeneChip

®

(Affymetrix, Inc.); Agilent

®

(Agilent

Technologies, Inc.); Applied Biosystems

®

(Applera Corporation or its subsidiaries); Eppendorf

®

(Eppendorf AG); Rotor-Gene

®

(Corbett Research Pty Ltd);

Ovation

®

(NuGEN Technologies, Inc.); SYBR

®

(Molecular Probes, Inc.).

Limited License Agreement

Use of this product signifies the agreement of any purchaser or user of the PAXgene Blood miRNA Kit to the following terms:

1. The PAXgene Blood miRNA Kit may be used solely in accordance with the PAXgene Blood miRNA Kit Handbook and for use with components contained in the Kit only. PreAnalytiX grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the PAXgene Blood miRNA Kit Handbook and additional protocols available at www.preanalytix.com.

2. Other than expressly stated licenses, PreAnalytiX makes no warranty that this Kit and/or its use(s) do not infringe the rights of third-parties.

3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.

4. PreAnalytiX specifically disclaims any other licenses, expressed or implied other than those expressly stated.

5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. PreAnalytiX may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and/or its components.

For updated license terms, see www.preanalytix.com.

© 2009 PreAnalytiX GmbH, all rights reserved.

PreAnalytiX

PreAnalytiX GmbH

Feldbachstrasse

CH – 8634 Hombrechtikon

Switzerland

PreAnalytiX Distributors

PreAnalytiX products are manufactured for PreAnalytiX by QIAGEN or BD and are distributed for PreAnalytiX by QIAGEN or BD. Products cannot be ordered at

PreAnalytiX GmbH.

Please see pages 42–43 for contact information for your local PreAnalytiX distributor.

Contents

Kit Contents

Shipping and Storage

Product Use Limitations

Product Warranty and Satisfaction Guarantee

Quality Control

Technical Assistance

Safety Information

Introduction

Principle and procedure

Equipment and Reagents to Be Supplied by User

Protocols

Manual Purification of Total RNA, Including miRNA, from Human

Whole Blood Collected into PAXgene Blood RNA Tubes

Automated Purification of Total RNA, Including miRNA, from

Human Whole Blood Collected into PAXgene Blood RNA Tubes

Troubleshooting Guide

Appendix A: General Remarks on Handling RNA

Appendix B: Storage, Quantification, and Determination of Quality of RNA

Appendix C: Using the QIAcube

Ordering Information

PreAnalytiX Worldwide

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PAXgene Blood miRNA Kit Handbook 05/2009

3

4

Kit Contents

PAXgene Blood miRNA Kit

Catalog no.

Number of preps

PAXgene RNA Spin Columns (red)

PAXgene Shredder Spin Columns (lilac)

Microcentrifuge Tubes (1.5 ml)

Processing Tubes (2 ml)

Buffer BM1 (resuspension buffer)

Buffer BM2* (binding buffer)

Buffer BM3* † (wash buffer concentrate)

Buffer BM4

†

(wash buffer concentrate)

Buffer BR5 (elution buffer)

RNase-Free Water

Proteinase K

RNase-Free DNase Set:

• RNase-Free DNase I (lyophilized)

• Buffer RDD (DNA digestion buffer)

• RNase-Free Water

Secondary Hemogard Closures

Handbook

(50)

763134

50

50

50

3 x 50

1 x 10

6 x 50

20 ml

18 ml

15 ml

11 ml

6 ml

2 x 125 ml

2 x 1.4 ml

1500 units ‡

2 x 2 ml

1.5 ml

50

1

* Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety information.

† Before using for the first time, add the required volume of ethanol (96–100%) as indicated on the bottle to obtain a working solution.

‡ Kunitz units are the commonly used units for measuring DNase I; see page 15 for definition.


Shipping and Storage

The PAXgene Blood miRNA Kit is shipped at ambient temperature. The RNase-Free

DNase Set box (containing RNase-free DNase, Buffer RDD, and RNase-free water) should be stored immediately upon receipt at 2–8°C. The remaining components of the kits should be stored dry at room temperature (15–25°C). All kit components are stable for at least 9 months under these conditions.

Product Use Limitations

For Research Use Only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The performance characteristics of this product have not been fully established.

All due care and attention should be exercised in the handling of the products. We recommend all users of PreAnalytiX ® products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines.

Product Warranty and Satisfaction Guarantee

PreAnalytiX guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, PreAnalytiX will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a PreAnalytiX product does not meet your expectations, simply call your local QIAGEN Technical Service Department or distributor. We will credit your account or exchange the product — as you wish. Separate conditions apply to scientific instruments, service products, and to products shipped on dry ice. Please inquire for more information.

A copy of PreAnalytiX terms and conditions can be obtained on request, and is also provided on the back of our invoices. If you have questions about product specifications or performance, please call QIAGEN Technical Services or your local distributor

(see page 42 or visit www.qiagen.com).

Quality Control

In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of

PAXgene Blood miRNA Kit is tested against predetermined specifications to ensure consistent product quality.


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6

Technical Assistance

At QIAGEN, we pride ourselves on the quality and availability of our technical support.

Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of

QIAGEN products. If you have any questions or experience any difficulties regarding the PAXgene Blood miRNA Kit or PreAnalytiX products in general, please do not hesitate to contact us.

PreAnalytiX customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at PreAnalytiX. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques.

For technical assistance and more information, please see our Technical Support Center at www.qiagen.com/Support or call one of the QIAGEN Technical Service

Departments or local distributors (see page 42 or visit www.qiagen.com).

Safety Information

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at www.preanalytix.com/rna_msds.asp where you can find, view, and print the

MSDS for each PreAnalytiX kit and kit component.

CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.

Buffer BM2 and Buffer BM3 contain guanidine thiocyanate. Guanidine salts can form highly reactive compounds when combined with bleach. If liquid containing these buffers is spilt, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.

The following risk and safety phrases apply to the components of the PAXgene Blood miRNA Kit.


Buffer BM2 and Buffer BM3

Xn

Contains guanidine thiocyanate: harmful. Risk and safety phrases:* R20/21/22-32,

S13-26-36-46

Proteinase K

Xn

Contains proteinase K: sensitizer, irritant. Risk and safety phrases:* R36/37/38-

42/43, S23-24-26-36/37

RNase-free DNase I

Xn

Contains deoxyribonuclease: sensitizer. Risk and safety phrases:* R42/43, S22-24-26-

36/37

24-hour emergency information

Emergency medical information in English, French, and German can be obtained

24 hours a day from:

Poison Information Center Mainz, Germany

Tel: +49-6131-19240

* R20/21/22: Harmful by inhalation, in contact with skin and if swallowed; R32: Contact with acids liberates very toxic gas; R36/37/38: Irritating to eyes, respiratory system and skin; R42/43: May cause sensitization by inhalation and skin contact; S13: Keep away from food, drink and animal feeding stuffs;

S22: Do not breathe dust; S23: Do not breathe vapor; S24: Avoid contact with skin; S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice; S36: Wear suitable protective clothing; S36/37: Wear suitable protective clothing and gloves; S46: If swallowed, seek medical advice immediately and show the container or label.


7

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Introduction

The PAXgene Blood miRNA System provides a complete solution for stabilization and purification of high-quality total RNA >18 nucleotides (including miRNA) from human whole blood. Blood is collected and stabilized in PAXgene Blood RNA Tubes. Total

RNA >18 nucleotides (including miRNA) is then purified using the PAXgene Blood miRNA Kit.

The purified total RNA is ready to use and is ideally suited for any downstream application, including:

• RT-PCR and real-time RT-PCR

• Differential display

• cDNA synthesis

• Northern, dot, and slot blot analyses

• Primer extension

• Poly A + RNA selection

• RNase/S1 nuclease protection

• Microarrays

The PAXgene Blood miRNA Kit allows the parallel processing of multiple samples either manually or on the QIAcube ® . The PAXgene Blood miRNA procedure replaces traditional methods involving the use of toxic substances such as phenol and/or chloroform, or time-consuming and tedious methods such as alcohol precipitation.

Important: The PAXgene Blood miRNA Kit can only be used in conjunction with

PAXgene Blood RNA Tubes. The tubes are not included in the kits and are available from BD and BD authorized distributors (cat. no. 762165, see page 40 for ordering information).

Principle and procedure

Blood samples are collected in PAXgene Blood RNA Tubes, which contain a reagent that lyses blood cells and immediately stabilizes intracellular RNA to preserve the gene expression profile. RNA stabilization is critical for reliable downstream gene expression analysis. Without stabilization, degradation of RNA and upregulation or downregulation of transcripts can occur immediately after blood is drawn. Blood samples collected in PAXgene Blood RNA Tubes can be safely stored or transported at

15–25°C for up to 72 hours, at 2–8°C for up to 5 days, or at –20°C or –70°C for at least 50 months without showing any significant RNA degradation or changes in transcript levels.


Total RNA >18 nucleotides (including miRNA) is purified from the stabilized blood samples using well-established PAXgene silica-membrane technology. PAXgene Blood

RNA Tubes are first centrifuged to pellet the samples, which are then washed with water and resuspended in Buffer BM1. After digestion in Buffer BM2 with proteinase K, the samples are homogenized by centrifugation through PAXgene Shredder spin columns.

Isopropanol is added to the samples to optimize binding conditions, and the samples are then centrifuged through PAXgene RNA spin columns, where total RNA

>18 nucleotides (including miRNA) binds to the PAXgene silica-membrane. The bound

RNA is subjected to DNase digestion to remove genomic DNA contamination and washed with Buffer BM3 followed by Buffer BM4. Pure RNA is then eluted in Buffer BR5.

With the PAXgene Blood miRNA Kit, all RNA molecules longer than 18 nucleotides are purified. The purified RNA includes both mRNA and small RNAs such as miRNA.


9

10

Blood

The Manual PAXgene Blood miRNA Procedure

Add proteinase K and Buffer BM2

Incubate

Transfer to PAXgene

Shredder Spin Column

Mix

Transfer supernatant of flow-through to microcentrifuge tube

Add isopropanol

Wash pellet

Load on PAXgene RNA

Spin Column

Bind total RNA

Total RNA >18 nt (includes miRNA)

Wash with Buffer BM3

Digest DNA

Resuspend in

Buffer BM1

Transfer to microcentrifuge tube

Wash with Buffer BM3 and Buffer BM4

Elute with Buffer BR5

Heat to 65°C

Ready-to-use RNA >18 nt (includes miRNA)


The Automated PAXgene Blood miRNA Procedure

Blood

Add proteinase K and Buffer BM2

Incubate

Mix

Transfer to

PAXgene Shredder spin column

Wash pellet

Add isopropanol to flow-through

Load on PAXgene

RNA Spin Column

Bind total RNA >18 nt

(includes miRNA)

Wash with Buffer BM3

Resuspend in

Buffer BM1

Transfer to processing tube and load into the

QIAcube shaker

Digest DNA

Wash with Buffer BM3 and Buffer BM4

Transfer PAXgene

RNA Spin Column

Elute

Close lids of microcentrifuge tubes and transfer to

QIAcube shaker

Heat to 65°C

Ready-to-use RNA >18 nt (includes miRNA)


11

Equipment and Reagents to Be Supplied by User

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.

For all protocols

• PAXgene Blood RNA Tubes (BD and BD authorized distributors, cat. no. 762165)

• Ethanol (96–100%, purity grade p.a.)*

• Isopropanol (100%, purity grade p.a.)

• Pipets (10 µl – 4ml) †

• Sterile, aerosol-barrier, RNase-free pipet tips

• Graduated cylinder

• Centrifuge† capable of attaining 1000–8000 x g, and equipped with a swingout rotor and buckets to hold PAXgene Blood RNA Tubes

• Vortex mixer †

• Crushed ice

• Permanent pen for labeling

For the manual protocol

• Variable speed microcentrifuge † capable of attaining 1000–8000 x g, and equipped with a rotor for 2 ml microcentrifuge tubes

• Shaker–incubator † capable of incubating at 55°C and 65°C and shaking at 400 rpm, but not exceeding 1400 rpm (e.g., Eppendorf ® Thermomixer Compact ‡ or equivalent)

* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.

† Ensure that instruments have been checked, maintained, and calibrated regularly according to the manufacturer’s recommendations.

‡ This is not a complete list of suppliers and does not include many important vendors of biological supplies.

12


For the automated protocol

• QIAcube* (QIAGEN, cat. no. 9001292 [110 V], cat. no. 9001293 [230 V])

• QIAcube consumables

• Filter-Tips, 1000 µl (1024) (QIAGEN, cat. no. 990352) †

• Reagent Bottles, 30 ml (6) (QIAGEN, cat. no. 990393) †

• Rotor Adapters (10 x 24) (QIAGEN, cat. no. 990394) †

• QIAcube accessories

• Reagent Bottle Rack (QIAGEN, cat. no. 990390) †

• Rotor Adapter Holder (QIAGEN, cat. no. 990392) †

• Scissors

* Ensure that instruments have been checked, maintained, and calibrated regularly according to the manufacturer’s recommendations.

† Also included in the Starter Pack, QIAcube (QIAGEN, cat. no. 990395).


13

Protocol: Manual Purification of Total RNA, Including miRNA, from Human Whole Blood Collected into

PAXgene Blood RNA Tubes

Important points before starting

• When using a pipet, ensure that it is set to the correct volume, and that liquid is carefully and completely aspirated and dispensed.

• To avoid transferring samples to the wrong tubes and plastic consumables, ensure that all processing tubes, microcentrifuge tubes, and rotor adapters are properly labeled using a permanent pen. Label the lid and the body of each microcentrifuge tube, the body of each processing tube, and the outer wall of each rotor adapter.

• Spillages of samples and buffers during the procedure may reduce the yield and purity of RNA.

• Unless otherwise indicated, all steps of this protocol, including centrifugation steps, should be carried out at room temperature (15–25°C).

• Because of the sensitivity of nucleic acid amplification technologies, the following precautions are necessary when handling samples to avoid cross-contamination:

• Carefully pipet the sample into the bottom of the tube without moistening the rim of the tube.

• Always change pipet tips between liquid transfers. Use aerosol-barrier pipet tips.

• Avoid touching the spin column membrane with the pipet tip.

• After vortexing or heating a microcentrifuge tube, briefly centrifuge it to remove drops from the inside of the lid.

• Wear gloves throughout the entire procedure. In case of contact between gloves and sample, change gloves immediately.

Things to do before starting

• Blood must be collected in PAXgene Blood RNA Tubes according to the instructions in the

PAXgene Blood RNA Tube Product Circular.

• Ensure that the PAXgene Blood RNA Tubes are incubated for at least 2 hours at room temperature after blood collection to ensure complete lysis of blood cells.

Incubation of the PAXgene Blood RNA Tube overnight may increase yields. If the

PAXgene Blood RNA Tube was stored at 2–8°C, –20°C, or –70°C after blood collection, first equilibrate it to room temperature, and then store it at room temperature for 2 hours before starting the procedure.

14


• Read the safety information on page 6.

• Read the guidelines on handling RNA (Appendix A, page 26).

• Ensure that instruments such as pipets have been checked and calibrated regularly according to the manufacturer’s recommendations.

• Buffers BM2 and BM3 may form a precipitate upon storage. If necessary, warm to 37°C to dissolve.

• Buffers BM3 and BM4 are supplied as a concentrate. Before using for the first time, add the appropriate volume of ethanol (96–100%, purity grade p.a.) as indicated on the bottle to obtain a working solution.

• If using the RNase-Free DNase Set for the first time, prepare DNase I stock solution.

Dissolve the solid DNase I (1500 Kunitz units)* in 550 µl of RNase-free water provided with the set. Take care that no DNase I is lost when opening the vial. Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the vial.

• Current data show that reconstituted DNase I can be stored at 2–8°C for up to

6 weeks. For long-term storage of DNase I, remove the stock solution from the glass vial, divide it into single-use aliquots (use the 1.5 ml microcentrifuge tubes supplied with the kit; there are enough for 5 aliquots), and store at –20°C for up to

9 months. Thawed aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.

• When reconstituting and aliquoting DNase I, ensure that you follow the guidelines for handling RNA (Appendix A, page 26).

Procedure

1.

Centrifuge the PAXgene Blood RNA Tube for 10 min at 3000–5000 x g using a swing-out rotor.

Note: Ensure that the blood sample has been incubated in the PAXgene Blood RNA

Tube for a minimum of 2 h at room temperature (15–25°C), in order to achieve complete lysis of blood cells.

Note: The rotor must contain tube adapters for round-bottom tubes. If other types of tube adapter are used, the tubes may beak during centrifugation.

2.

Remove the supernatant by decanting or pipetting. Add 4 ml RNase-free water to the pellet, and close the tube using a fresh secondary Hemogard closure.

If decanting the supernatant, take care not to disturb the pellet, and dry the rim of the tube with a clean paper towel.

* Kunitz units are the commonly used units for measuring DNase I, defined as the amount of DNase I that causes an increase in A

260 of 0.001 per minute per milliliter at 25°C, pH 5.0, with highly polymerized DNA as the substrate (Kunitz, M. (1950) J. Gen. Physiol. 33, 349 and 363).


15

3.

Vortex until the pellet is visibly dissolved, and centrifuge for 10 min at

3000–5000 x g using a swing-out rotor. Remove the entire supernatant by decanting or pipetting, and discard.

Small debris remaining in the supernatant after vortexing but before centrifugation will not affect the procedure.

Note: Incomplete removal of the supernatant will inhibit lysis and dilute the lysate, and therefore affect the conditions for binding RNA to the PAXgene membrane.

4.

Add 350 µl Buffer BM1, and vortex until the pellet is visibly dissolved.

5.

Pipet the sample into a 1.5 ml microcentrifuge tube. Add 300 µl Buffer BM2 and

40 µl proteinase K. Mix by vortexing for 5 s, and incubate for 10 min at 55°C in a shaker–incubator at 400–1400 rpm. After incubation, set the temperature of the shaker–incubator to 65°C for use in step 20.

Note: Do not mix Buffer BM2 and proteinase K together before adding them to the sample.

6.

Pipet the sample into a PAXgene Shredder spin column (lilac) placed in a 2 ml processing tube, and centrifuge for 3 min at full speed (do not exceed 20,000 x g).

7.

Carefully transfer the entire supernatant of the flow-through from the PAXgene

Shredder spin column to a new 1.5 ml microcentrifuge tube without disturbing the pellet in the processing tube.

8.

Add 700 µl of isopropanol (100%, purity grade p.a.), and mix by vortexing.

9.

Pipet 700 µl sample into the PAXgene RNA spin column (red) placed in a 2 ml processing tube. Close the lid gently, and centrifuge for 1 min at 8000–20,000 x g.

Place the spin column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.*

10. Pipet the remaining sample into the PAXgene RNA spin column (red). Close the lid gently, and centrifuge for 1 min at 8000–20,000 x g. Place the spin column in a new

2 ml processing tube, and discard the old processing tube containing flow-through.*

11. Add 350 µl Buffer BM3 to the PAXgene RNA spin column. Close the lid gently, and centrifuge for 15 s at 8000–20,000 x g. Place the spin column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.*

Note: Buffer BM3 is supplied as a concentrate. Ensure that ethanol is added to

Buffer BM3 before use (see “Things to do before starting”).

* Flow-through contains Buffer BM2 or Buffer BM3 and is therefore not compatible with bleach. See page 6 for safety information.

16


12. Add 10 µl DNase I stock solution to 70 µl Buffer RDD in a 1.5 ml microcentrifuge tube.

Mix by gently flicking the tube, and centrifuge briefly to collect residual liquid from the sides of the tube.

Note: DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently flicking the tube. Do not vortex.

13. Pipet the DNase I incubation mix (80 µl) directly onto the PAXgene RNA spin column membrane, and incubate on the benchtop (20–30°C) for 15 min.

Note: Ensure that DNase I incubation mix is placed directly onto the membrane.

DNase digestion will be incomplete if part of the mix is applied to and remains on the walls or O-ring of the spin column.

14. Add 350 µl Buffer BM3 to the PAXgene RNA spin column. Close the lid gently, and centrifuge for 15 s at 8000–20,000 x g. Place the spin column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.*

15. Add 500 µl Buffer BM4 to the PAXgene RNA spin column. Close the lid gently, and centrifuge for 15 s at 8000–20,000 x g. Discard the flow-through. Place the spin column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.

Note: Buffer BM4 is supplied as a concentrate. Ensure that ethanol is added to

Buffer BM4 before use (see “Things to do before starting”).

16. Add another 500 µl Buffer BM4 to the PAXgene RNA spin column. Close the lid gently, and centrifuge for 2 min at 8000–20,000 x g.

Note: After centrifugation, carefully remove the PAXgene RNA spin column from the processing tube so that the column does not contact the flow-through.

Otherwise, carryover of ethanol will occur.

17. Discard the processing tube containing flow-through, and place the PAXgene RNA spin column in a new 2 ml processing tube (supplied). Centrifuge at 8000–20,000 x g for 1 min.

It is important to dry the spin column membrane, since residual ethanol may interfere with downstream reactions.

18. Discard the processing tube containing flow-through. Place the PAXgene RNA spin column in a new 1.5 ml microcentrifuge tube, and pipet 40 µl Buffer BR5 directly onto the spin column membrane. Close the lid gently, and centrifuge for 1 min at

8000–20,000 x g to elute the RNA.

Be sure to add Buffer BR5 directly to the spin column membrane. This wets the entire membrane, ensuring maximum elution efficiency.

19. Repeat the elution step (step 18) as described, using 40 µl Buffer BR5 and the same microcentrifuge tube.


17

20. Incubate the eluate for 5 min at 65°C in the shaker–incubator without shaking. After incubation, chill immediately on ice.

This incubation at 65°C denatures the RNA for downstream applications. Do not exceed the incubation time or temperature.

21. If the RNA eluate will not be used immediately, store at –20°C or –70°C. Since the

RNA remains denatured after freezing and thawing, it is not necessary to repeat the incubation at 65°C.

Note: If quantifying the RNA using a spectrophotometer, refer to the guidelines in

Appendix A (page 26). For accurate quantification of RNA by absorbance at

260 nm, we recommend diluting the sample in 10 mM Tris Cl, pH 7.5. Dilution in RNase-free water may lead to inaccurately low values. Use Buffer BR5 to zero the spectrophotometer, and make sure to add the same volume of Buffer BR5 as the volume of eluted RNA to be diluted. Buffer BR5 shows high absorbance at

220 nm, which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed.

Note: For quantification in Tris buffer, use the relationship A

260

= 1 fi 44 µg/ml.

For RT-PCR and real-time RT-PCR with the purified RNA, QIAGEN offers a range of optimized, ready-to-use kits that provide highly specific and sensitive results. For details, visit www.qiagen.com/PCR.

18


Protocol: Automated Purification of Total RNA,

Including miRNA, from Human Whole Blood Collected into PAXgene Blood RNA Tubes

Important points before starting

• When using a pipet, ensure that it is set to the correct volume, and that liquid is carefully and completely aspirated and dispensed.

• To avoid transferring samples to the wrong tubes and plastic consumables, ensure that all processing tubes, microcentrifuge tubes, and rotor adapters are properly labeled using a permanent pen. Label the lid and the body of each microcentrifuge tube, the body of each processing tube, and the outer wall of each rotor adapter.

• Spillages of samples and buffers during the procedure may reduce the yield and purity of RNA.

• Unless otherwise indicated, all steps of this protocol, including centrifugation steps, should be carried out at room temperature (15–25°C).

• Because of the sensitivity of nucleic acid amplification technologies, the following precautions are necessary when handling samples to avoid cross-contamination:

• Carefully pipet the sample into the bottom of the tube without moistening the rim of the tube.

• Always change pipet tips between liquid transfers. Use aerosol-barrier pipet tips.

• Avoid touching the spin column membrane with the pipet tip.

• After vortexing or heating a microcentrifuge tube, briefly centrifuge it to remove drops from the inside of the lid.

• Wear gloves throughout the entire procedure. In case of contact between gloves and sample, change gloves immediately.

Things to do before starting

• Blood must be collected in PAXgene Blood RNA Tubes according to the instructions in the PAXgene Blood RNA Tube Product Circular.

• Ensure that the PAXgene Blood RNA Tubes are incubated for at least 2 hours at room temperature after blood collection to ensure complete lysis of blood cells.

Incubation of the PAXgene Blood RNA Tube overnight may increase yields. If the

PAXgene Blood RNA Tube was stored at 2–8°C, –20°C, or –70°C after blood collection, first equilibrate it to room temperature, and then store it at room temperature for 2 hours before starting the procedure.


19

• Read the safety information on page 6.

• Read the guidelines on handling RNA (Appendix A, page 26).

• Read the QIAcube User Manual and any additional information supplied with the

QIAcube, paying careful attention to the safety information.

• Ensure that instruments, such as pipets and the QIAcube, have been checked and calibrated regularly according to the manufacturer’s recommendations.

• Buffers BM2 and BM3 may form a precipitate upon storage. If necessary, warm to 37°C to dissolve.

• Buffers BM3 and BM4 are supplied as a concentrate. Before using for the first time, add the appropriate volume of ethanol (96–100%, purity grade p.a.) as indicated on the bottle to obtain a working solution.

• If using the RNase-Free DNase Set for the first time, prepare DNase I stock solution.

Dissolve the solid DNase I (1500 Kunitz units)* in 550 µl of RNase-free water provided with the set. Take care that no DNase I is lost when opening the vial. Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the vial.

• Current data show that reconstituted DNase I can be stored at 2–8°C for up to

6 weeks. For long-term storage of DNase I, remove the stock solution from the glass vial, divide it into single-use aliquots (use the 1.5 ml microcentrifuge tubes supplied with the kit; there are enough for 5 aliquots), and store at–20°C for up to 9 months.

Thawed aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.

• When reconstituting and aliquoting DNase I, ensure that you follow the guidelines for handling RNA (Appendix A, page 26).

• Install the correct shaker adapter (included with the QIAcube; use the adapter for

2 ml safe-lock tubes, marked with a “2”), and place the shaker rack on top of the adapter.

• Check the waste drawer and empty it if necessary.

• Install the protocols if not already done for previous runs. Install both “PAXgene

Blood miRNA Part A” and “PAXgene Blood miRNA Part B” protocols. See

“Installing protocols on the QIAcube”, page 32.

* Kunitz units are the commonly used units for measuring DNase I, defined as the amount of DNase I that causes an increase in A

260 of 0.001 per minute per milliliter at 25°C, pH 5.0, with highly polymerized

DNA as the substrate (Kunitz, M. (1950) J. Gen. Physiol. 33, 349 and 363).

20


Procedure

1.

Close the QIAcube door, and switch on the QIAcube with the power switch (see

Figure 1, page 31).

A beeper sounds and the startup screen appears. The instrument automatically performs initialization tests.

2.

Open the QIAcube door, and load the necessary reagents and plasticware into the

QIAcube. See “Loading the QIAcube”, pages 32–38.

To save time, loading can be performed during one or both of the following

10-minute centrifugation steps (steps 3 and 5).

3.

Centrifuge the PAXgene Blood RNA Tube for 10 min at 3000–5000 x g using a swing-out rotor.

Note: Ensure that the blood sample has been incubated in the PAXgene Blood RNA

Tube for a minimum of 2 h at room temperature (15–25°C), in order to achieve complete lysis of blood cells.

Note: The rotor must contain tube adapters for round-bottom tubes. If other types of tube adapter are used, the tubes may beak during centrifugation.

4.

Remove the supernatant by decanting or pipetting. Add 4 ml RNase-free water to the pellet, and close the tube using a fresh secondary BD Hemogard closure (supplied with the kit).

If the supernatant is decanted, take care not to disturb the pellet, and dry the rim of the tube with a clean paper towel.

5.

Vortex until the pellet is visibly dissolved, and centrifuge for 10 min at

3000–5000 x g using a swing-out rotor. Remove and discard the entire supernatant.

Small debris remaining in the supernatant after vortexing but before centrifugation will not affect the procedure.

Note: Incomplete removal of the supernatant will inhibit lysis and dilute the lysate, and therefore affect the conditions for binding RNA to the PAXgene membrane.

6.

Add 350 µl Buffer BM1, and vortex until the pellet is visibly dissolved.

7.

Pipet the sample into a 2 ml processing tube.

Note: Use the 2 ml processing tubes included in the PAXgene Blood miRNA Kit.

8.

Load the open processing tubes containing sample into the QIAcube shaker (see

Figure 3, page 34, and Figure 7, page 37). The sample positions are numbered for ease of loading. Insert shaker rack plugs (included with the QIAcube) into the slots at the edge of the shaker rack next to each processing tube. This enables detection of samples during the load check.

Note: Make sure that the correct shaker adapter (Shaker Adapter, 2 ml, safe-lock tubes, marked with a “2”, included with the QIAcube) is installed.


21

Note: If processing fewer than 12 samples, make sure to load the shaker rack as shown in Figure 7, page 37. One or 11 samples cannot be processed. The position numbers in the shaker rack correspond to the position numbers in the centrifuge.

9.

Close the QIAcube instrument door (see Figure 1, page 31).

10. Select the “PAXgene Blood miRNA Part A” protocol, and start the protocol.

Follow the instructions given on the QIAcube touchscreen.

Note: Make sure that both program parts (part A and part B) are installed on the

QIAcube instrument (see “Installing protocols on the QIAcube”, page 32).

Note: The QIAcube will perform load checks for samples, tips, rotor adapters, and reagent bottles.

11. After the “PAXgene Blood miRNA Part A” protocol is finished, open the QIAcube instrument door (see Figure 1, page 31). Remove and discard the PAXgene RNA spin columns from the rotor adapters and the empty processing tubes from the shaker.

Note: During the run, spin columns are transferred from the rotor adapter position 1

(lid position L1) to rotor adapter position 3 (lid position L2) by the instrument (see

Figure 5, page 35).

12. Close the lids of all 1.5 ml microcentrifuge tubes containing the purified RNA in the rotor adapters (position 3, lid position L3, see Figure 5, page 35). Transfer the 1.5 ml microcentrifuge tubes onto the QIAcube shaker adapter (see Figure 3, page 34, and


13. Close the QIAcube instrument door (see Figure 1, page 31).

14. Select the “PAXgene Blood miRNA Part B” protocol, and start the protocol.

Follow the instructions given on the QIAcube touchscreen.

Note: This program incubates the samples at 65°C and denatures the RNA for downstream applications. Even if the downstream application includes a heat denaturation step, do not omit this step. Sufficient RNA denaturation is essential for maximum efficiency in downstream applications.

15. After the “PAXgene Blood miRNA Part B” program is finished, open the QIAcube instrument door (see Figure 1, page 31). Immediately place the microcentrifuge tubes containing the purified RNA on ice.

WARNING Hot surface

The shaker can reach temperatures of up to 70°C (158°F).

Avoid touching it when it is hot.

Note: Do not let the purified RNA remain in the QIAcube. Since the samples are not cooled, the purified RNA can be degraded. Unattended overnight sample preparation runs are therefore not recommended.

22


16. If the RNA samples will not be used immediately, store at –20°C or –70°C.

Since the RNA remains denatured after repeated freezing and thawing, it is not necessary to repeat the heat incubation protocol (“PAXgene Blood miRNA Part B”).

Note: If quantifying the RNA using a spectrophotometer, refer to the guidelines in

Appendix A (page 26). For accurate quantification of RNA by absorbance at

260 nm, we recommend diluting the sample in 10 mM Tris Cl, pH 7.5.* Dilution in

RNase-free water may lead to inaccurately low values. Use Buffer BR5 to zero the spectrophotometer, and make sure to add the same volume of Buffer BR5 as the volume of eluted RNA to be diluted. Buffer BR5 shows high absorbance at 220 nm, which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed.

Note: For quantification in Tris buffer, use the relationship A

260

= 1 fi 44 µg/ml.

For RT-PCR and real-time RT-PCR with the purified RNA, QIAGEN offers a range of optimized, ready-to-use kits that provide highly specific and sensitive results. For details, visit www.qiagen.com/PCR.

17. Remove the reagent bottle rack from QIAcube worktable (see Figure 3, page 34), and close all bottles with the appropriately labeled lids. Buffer in bottles can be stored at room temperature (15–25°C) for up to 3 months. Remove and discard remaining reagents in the processing tubes in the QIAcube microcentrifuge tube slots (see Figure

3, page 34). Remove and discard rotor adapters from the centrifuge (see Figure 3, page 34). Empty the QIAcube waste drawer (see Figure 1, page 31). Close the

QIAcube instrument door, and switch off the instrument with the power switch (see


* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.

For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.


23

Troubleshooting Guide

This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical

Support Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN

Technical Services are always happy to answer any questions you may have about either the information or protocols in this handbook or sample and assay technologies

(for contact information, see page 42 or visit www.qiagen.com).

Comments and suggestions

Low RNA yield

a) Less than 2.5 ml blood Ensure that 2.5 ml blood is collected in the collected in PAXgene Blood PAXgene Blood RNA Tube (see

PAXgene Blood

RNA Tube

RNA Tube Product Circular).

b) RNA diluted in water for

A

260 measurement

For accurate quantification, RNA must be diluted in 10 mM Tris·Cl, pH 7.5 (see Appendix B, page 28).

c) RNA still bound to PAXgene Repeat RNA elution, but incubate the PAXgene

RNA spin column membrane RNA spin column on the benchtop for 10 min with

Buffer BR5 before centrifuging.

d) Cell debris transferred to

PAXgene RNA spin column

Be sure to vortex the pellet at step 3 of the manual protocol or step 5 of the automated protocol

(however, the presence of small debris does not affect the procedure).

e) Supernatant not completely Be sure to remove the entire supernatant. If removed after centrifuging decanting the supernatant, remove drops from

PAXgene Blood RNA Tube the rim of the tube by dabbing onto a paper towel. f) Blood incubated for less than After collecting blood in PAXgene Blood RNA

2 h in PAXgene Blood RNA Tubes, be sure to incubate for at least 2 h at room

Tubes temperature (15–25°C).

g) Manual protocol: General yield is good, but miRNA content is low

Ensure that the sample and the isopropanol in step 8 of the manual protocol are mixed completely until no phase separation is visible.

24


Comments and suggestions

Low A

260

/ A

280 value

a) Water used to dilute RNA for Use 10 mM Tris·Cl, pH 7.5, not RNase-free water,

A

260

/

A

280 measurement to dilute the sample before measuring purity (see

Appendix B, page 28) b) Spectrophotometer not properly zeroed

To zero the spectrophotometer, use a blank containing the portion of Buffer BR5 and dilution buffer as in the samples to be measured. Buffer

BR5 shows high absorbance at 220 nm, which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed.

RNA degraded

RNase contamination Although all PAXgene buffers have been tested and are guaranteed RNase-free, RNases can be introduced during use. Be certain not to introduce any RNases during the PAXgene RNA procedure or later handling. See Appendix A (page 26) for general remarks on handling RNA.

Do not put RNA samples into a vacuum dryer or microcentrifuge that has been used in DNA preparation where RNases may have been used.

RNA does not perform well in downstream experiments

High levels of globin mRNA High levels of globin mRNA in purified RNA from affect microarray analysis whole blood reduce the sensitivity of gene expression analysis with Affymetrix ® GeneChip ® arrays. To avoid this problem, we recommend preparing targets for GeneChip arrays using the

Ovation ® Whole Blood Solution from NuGEN

(www.nugeninc.com).

Instrument malfunction

QIAcube not operated properly

Read the

QIAcube User Manual, paying careful attention to the Troubleshooting section. Make sure that the QIAcube is properly maintained, as described in the

QIAcube User Manual.


25

Appendix A: General Remarks on Handling RNA

Handling RNA

Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA, do not use any plasticware or glassware without first eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. In order to create and maintain an RNase-free environment, the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA.

General handling

Proper microbiological, aseptic technique should always be used when working with

RNA. Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed whenever possible. Keep purified RNA on ice when aliquots are pipetted for downstream applications.

Disposable plasticware

The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases.

Nondisposable plasticware

Nondisposable plasticware should be treated before use to ensure that it is RNasefree. Plasticware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA* followed by RNase-free water (see ”Solutions”, page 27). Alternatively, chloroformresistant plasticware can be rinsed with chloroform* to inactivate RNases.



26


Glassware

Glassware should be treated before use to ensure that it is RNase-free. Glassware used for RNA work should be cleaned with a detergent,* thoroughly rinsed, and oven baked at 240°C for at least 4 hours (overnight, if more convenient) before use.

Autoclaving alone will not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC* (diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water), allow to stand overnight (12 hours) at 37°C, and then autoclave or heat to

100°C for 15 minutes to eliminate residual DEPC.

Electrophoresis tanks

Electrophoresis tanks should be cleaned with detergent solution (e.g., 0.5% SDS),* thoroughly rinsed with RNase-free water, and then rinsed with ethanol † and allowed to dry.

Solutions

Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of 0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to

100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove any trace of DEPC. DEPC will react with primary amines and cannot be used directly to treat Tris* buffers. DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO

2

. When preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation.

Carbethoxylated RNA is translated with very low efficiency in cell-free systems.

However, its ability to form DNA:RNA or RNA:RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified. Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100°C for

15 minutes.

Note: PAXgene RNA buffers are guaranteed RNase-free without using DEPC treatment and are therefore free of any DEPC contamination.



† Plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the supplier’s instructions.


27

Appendix B: Storage, Quantification, and

Determination of Quality of RNA

Storage of RNA

Purified RNA may be stored at –20°C or –70°C in Buffer BR5. Under these conditions, no degradation of RNA is detectable after 1 year.

Quantification of miRNA

Since the RNA eluate obtained using this procedure is enriched in various small RNA species, the yield of specific small RNA species (e.g., miRNA) cannot be quantified by

OD measurement or fluorogenic assays. Instead, we recommend using quantitative, real-time RT-PCR assays, such as the miScript PCR System, specific for the type of small

RNA under study. For example, to estimate miRNA yield, an assay directed against any miRNA known to be adequately expressed in the samples being processed may be used.

The miScript PCR System is a three-component system that covers all the steps of conversion of miRNA and mRNA into cDNA and detection of miRNAs in SYBR ® Greenbased real-time PCR. A single cDNA synthesis reaction is sufficient for analysis of multiple miRNAs. The miScript PCR System can also be used for detection of other small RNAs, such as snoRNAs or piRNAs. See page 41 for ordering information.

Quantification of RNA

The concentration of RNA can be determined by measuring the absorbance at 260 nm

( A

260

) in a spectrophotometer (see “Spectrophotometric quantification of RNA” below).

For small amounts of RNA, however, it may not be possible to accurately determine amounts photometrically. Small amounts of RNA can be quantified using the QIAxcel ® system (www.qiagen.com/QIAxcel) or Agilent ® 2100 bioanalyzer, fluorometric quantification, or quantitative, real-time RT-PCR.

Spectrophotometric quantification of RNA

To ensure significance, A

260 readings should be greater than 0.15. An absorbance of

1 unit at 260 nm corresponds to 44 µg of RNA per ml ( A

260

=1 fi 44 µg/ml). This relation is valid only for measurements at a neutral pH. Therefore, if it is necessary to dilute the RNA sample, this should be done in a buffer with neutral pH.* As discussed below (see “Purity of RNA”, page 29), the ratio between the absorbance values at

260 and 280 nm gives an estimate of RNA purity.



28


When measuring RNA samples, be certain that cuvettes are RNase-free, especially if the RNA is to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1 M NaOH, 1 mM EDTA* followed by washing with RNasefree water (see “Solutions”, page 27). When zeroing the spectrophotometer, be sure to use a solution that contains Buffer BM5 at the same dilution as the RNA solution to be quantified. An example of the calculation involved in RNA quantification is shown below:

Volume of RNA sample = 80 ml

Dilution = 10 ml of RNA sample + 140 ml 10 mM Tris·Cl, pH 7.5 (1/15 dilution)

Measure absorbance of diluted sample in a cuvette (RNase-free).

A

260

= 0.3

Concentration of RNA sample = 44 ml/ml x A

260 x dilution factor

= 44 ml/ml x 0.3 x 15

= 198 mg/ml

Total yield = concentration x volume of sample in milliliters

= 198 mg/ml x 0.08 ml

= 15.8 mg RNA

Purity of RNA

The ratio of the readings at 260 nm and 280 nm ( A

260

/ A

280

) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum, such as protein. However, the A

260

/ A

280 ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A

260 results in a lower A

260

/ A

280

/ A

280 ratio can vary greatly. Lower pH ratio and reduced sensitivity to protein contamination. For accurate values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5.

Pure RNA has an A

260

/ A

280 ratio of 1.9–2.1

† in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer with the same solution used for dilution.

For determination of RNA concentration, however, we recommend dilution of the sample in a buffer with neutral pH since the relationship between absorbance and concentration (

A

260 reading of 1 = 44 µg/ml RNA) is based on an extinction coefficient calculated for RNA at neutral pH (see “Quantification of RNA”, page 28).

† Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some spectrophotometers.


29

DNA contamination

No currently available purification method can guarantee that RNA is completely free of DNA, even when it is not visible on an agarose gel. While the vast majority of cellular DNA will by removed by the DNase digestion step, trace amounts may still remain in the purified RNA.

For analysis of very low abundance targets, any interference by residual DNA contamination can be detected by performing real-time RT-PCR control experiments in which no reverse transcriptase is added prior to the PCR step.

To prevent any interference by DNA in real-time RT-PCR applications, such as with

Rotor-Gene ® Q and Applied Biosystems ® instruments, we recommend designing primers that anneal at intron splice junctions so that genomic DNA will not be amplified. QuantiTect ® Primer Assays from QIAGEN (www.qiagen.com/GeneGlobe) are designed for SYBR Green–based real-time RT-PCR analysis of RNA sequences

(without detection of genomic DNA) where possible. For real-time RT-PCR assays where amplification of genomic DNA cannot be avoided, the QuantiTect Reverse

Transcription Kit provides fast cDNA synthesis with integrated removal of genomic

DNA contamination (see ordering information, page 41).

Integrity of RNA

The integrity and size distribution of total RNA purified with PAXgene Blood miRNA

Kit can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining* or by using the QIAxcel system or Agilent 2100 bioanalyzer. The respective ribosomal RNAs should appear as sharp bands or peaks. The apparent ratio of 28S rRNA to 18S rRNA should be approximately 2:1. In contrast to other RNA isolation procedures, ribosomal bands or peaks of a specific sample should be sharp and additionally a smear towards smaller sized RNAs should appear. This smear contains small RNA species, such as miRNA.



30


Appendix C: Using the QIAcube

Ensure that you are familiar with operating the QIAcube. Please read the QIAcube

User Manual and any additional information supplied with the QIAcube, paying careful attention to the safety information, before beginning the automated PAXgene

Blood miRNA protocol.

Starting the QIAcube

Close the QIAcube door, and switch on the QIAcube with the power switch (see

Figure 1).

A beeper sounds and the startup screen appears. The instrument automatically performs initialization tests.

Front View of the QIAcube

2

1 3 4

6

Figure 1

1

Touchscreen

2

Door

3

RS232 serial port behind protective panel (for use by QIAGEN Instrument Service Specialists only)

4

USB port behind protective panel

5

Power switch

6

Waste drawer


5

31

Installing protocols on the QIAcube

An initial protocol installation is required before the first RNA preparation run on the

QIAcube can be performed. Install both “PAXgene Blood miRNA Part A” and “PAXgene

Blood miRNA Part B” protocols.

Protocols are provided at www.qiagen.com/MyQIAcube and need to be downloaded to the USB stick supplied with the QIAcube and transferred to the QIAcube via the USB port. Save the files on the USB stick in a folder named New_Protocols.

The USB port, located behind the protective panel (see Figure 1), allows connection of the QIAcube to the USB stick supplied with the QIAcube. Data files, such as log files or report files can also be transferred via the USB port from the QIAcube to the USB stick.

Note: The USB port is only for use with the USB stick provided by QIAGEN. Do not connect other devices to this port.

Note: Do not remove the USB stick while downloading protocols or transferring data files or during a protocol run.

Loading the QIAcube

To save time, loading can be performed during one or both of the 10-minute centrifugation steps (steps 3 and 5) in “Protocol: Automated Purification of Total RNA,

Including miRNA, from Human Whole Blood Collected into PAXgene Blood RNA

Tubes”, page 21.

Reagent bottles

Carefully fill 4 QIAcube reagent bottles with the reagents listed in Table 1 (fill the bottles up to the indicator level on the reagent bottles). Label the bottles and lids clearly with buffer names and place into the appropriate position in the reagent bottle rack (see

Figures 2 and 3).

Before every run on the QIAcube, make sure that the reagent bottles are filled up to the indicator levels (remaining volumes in the original kit buffer bottles should be used to fill the reagent bottles). Position the reagent rack with filled reagent bottles onto the

QIAcube worktable as shown (Figures 2 and 3).

Note: Be sure to remove lids from the bottles before placing onto the worktable.

Note: Buffer volumes provided in the PAXgene Blood miRNA Kit are sufficient for a maximum of 7 RNA preparation runs on the QIAcube. Multiple runs with few samples should be avoided in order allow sufficient buffer volumes for processing the full

50 samples.

32


Table 1. Positions in the reagent bottle rack

4

5

2

3

6

Position

1

Reagent

Buffer BM2

100% isopropanol

Buffer BM3

Buffer BM4

– (leave empty)

– (leave empty)

* Buffer BM3 and Buffer BM4 are supplied as concentrates. Before using for the first time, add appropriate volumes of ethanol (96–100%, purity grade p.a.) as indicated on the bottle to obtain a working solution.

Loading the Reagent Bottle Rack

A

B

Figure 2

A

Schematic of positions and contents of bottles in the reagent bottle rack.

the QIAcube.

B

Loading the rack onto


33

Internal View of the QIAcube

1

9

2

5

8

6

7

3

4

Figure 3

1

Centrifuge lid

2

Centrifuge

3

Shaker

4

Reagent bottle rack

5

Tip sensor

6

Microcentrifuge tube slots

7

Tip racks

8

Disposal slots for tips and columns

9

Robotic arm

Spin columns, microcentrifuge tubes, and QIAcube plasticware

Place 2 tip racks filled with Filter-Tips 1000 µl onto the QIAcube (see Figure 3).

Refill racks with tips when necessary.

Note: Only use 1000 µl filter-tips designed for use with the QIAcube.

Label rotor adapters and microcentrifuge tubes for each sample using a permanent pen. Open the PAXgene Shredder spin columns to be used, and cut the lids off completely using scissors (see Figure 4).

Note: For proper operation of the QIAcube robotic gripper, completely remove (cut off) the lids and all plastic parts connecting the lid to the PAXgene Shredder spin columns

(see Figure 4). Otherwise, the robotic gripper cannot grip the spin columns properly.

34


Column lid removed correctly

Loading a PAXgene Shredder Spin Column

Column lid removed incorrectly; part of lid is remaining

Figure 4 The PAXgene Shredder spin column is loaded into the middle position of the rotor adapter. Cut off the lid before loading the column.

Load the PAXgene RNA spin column, PAXgene Shredder spin column (without lid), and labeled microcentrifuge tube into the appropriate positions in each labeled rotor adapter as shown in Table 2 and Figure 5.

Note: Make sure that the spin column and microcentrifuge tube lids are pushed all the way down to the bottom of the slots at the edge of the rotor adapter otherwise the lids will break off during centrifugation.

Table 2. Labware in the rotor adapter

2

3

Position

1

Reagent

PAXgene RNA spin column (red)

PAXgene Shredder spin column (lilac)*

Microcentrifuge tube †

†

* Cut off lid before placing in rotor adapter.

Use the microcentrifuge tubes (1.5 ml) included in the PAXgene Blood miRNA Kit.

Lid position

L1

–

L3

Positions in the Rotor Adapter

L2

L1

2

1 3

L3

Figure 5 The rotor adapter has three tube positions (1–3) and three lid positions (L1–L3).


35

Loading the centrifuge

Load the assembled rotor adapters into the centrifuge buckets as shown in Figure 6.

Note: If processing fewer than 12 samples, make sure to load the centrifuge rotor so that it is balanced radially (see Figure 7). All centrifuge buckets must be mounted before starting a protocol run, even if fewer than 12 samples are to be processed. A single

(one) sample or 11 samples cannot be processed.

Loading the Centrifuge

Figure 6 Load the assembled rotor adapters into the centrifuge buckets.

36


Loading the Centrifuge and Shaker

1 1

1 7 1

2 samples 3 samples

9

6 12

5

5

6

7

1

2

1

2

1

2

7

8

1

2

4 samples 5 samples

6 12

9

8

5 5

6

8

7

6 samples

9

8

7

1

2

3

1

2

3

6

7

8

9

12

7 samples

10

9

1

2

3

1

2

3

7

6

6

8 samples

10

9

8

1

2

3

4

3

4

1

2

6

7

7

8

9

10

12

9 samples

11

10

9

1

2

3

7

6

5

1

2

3

5

6

11

10 samples

10

9

8

1

2

3

5

4

3

4

1

2

5

6

7

8

9

10

11

12

7

Figure 7 Centrifuge and shaker positions are shown for processing from two to ten samples. One or

11 samples cannot be processed.

7

9

10

12

7

9

10

11

12

7

9

12

7

8

9

12


37

Processing tubes

Remove any processing tubes left in the microcentrifuge tube slots from previous runs

(see Figure 3, page 34). Fill 3 processing tubes with the amount of reagents given in

Table 3. Label the tubes clearly with reagent names and place them into the appropriate position in the microcentrifuge tube slots, as indicated in Table 4. Pipet the indicated volume of DNA digestion buffer (RDD) into a processing tube, and add the indicated volume of DNase I stock solution. Mix by gently pipetting the complete mixture up and down 3 times using a 1000 µl pipet tip. Use the 2 ml processing tubes included in the

PAXgene Blood miRNA Kit.

Note: Be sure to only pipet the required volume as indicated in Table 3.

Table 3. Volume of reagents required in processing tubes for the microcentrifuge tube slots

Volume of reagent required for the indicated number of samples (µl)

8

9

10

12

6

7

4

5

2

3

Number of samples Proteinase K

126

170

213

256

299

342

386

429

472

558

DNase I incubation mix

187 (23 DNase I + 164 Buffer RDD)










Buffer BR5

313

399

486

572

658

745

831

918

1004

1177

Table 4. Microcentrifuge tube slots

Content

Vessel

A

Proteinase K

Processing Tube*

Position

B

DNase I incubation mix

Processing Tube*

* Use the 2 ml processing tubes included in the PAXgene Blood miRNA Kit.

C

Elution buffer (BR5)

Processing Tube*

38



Product Contents Cat. no.

PAXgene Blood miRNA System

Products that can be ordered from QIAGEN

PAXgene Blood miRNA Kit (50) 50 PAXgene Spin Columns,

Processing Tubes, RNase-Free

DNase I, RNase-Free Reagents and

Buffers. To be used in conjunction with the PAXgene Blood RNA Tubes

QIAcube (110 V)*

QIAcube (230 V) †

Robotic workstation for automated purification of

DNA, RNA, or proteins using

QIAGEN spin-column kits,

1-year warranty on parts and labor ‡

763134

9001292*

9001293 †

Warranty PLUS 2 Full, QIAcube 3-year warranty, 48-hour

(2 working days) priority response, all labor, travel, and repair parts

Starter Pack, QIAcube Pack includes: reagent bottle racks (3); rack labeling strips (8);

200 µl filter-tips (1024); 1000 µl filter-tips (1024); 1000 µl filter-tips, wide-bore (1024); 30 ml reagent bottles (18); rotor adapters (240); rotor adapter holder

Filter-Tips, 1000 µl (1024)

Reagent Bottles, 30 ml (6)

Rotor Adapters (10 x 24)

Sterile, Disposable Filter-Tips, racked;

Reagent Bottles (30 ml) with lids; pack of 6; for use with the

QIAcube reagent bottle rack

For 240 preps: 240

Disposable Rotor Adapters; for use with the QIAcube

9240834

990395

990352

990393

990394

†

* US, Canada, and Japan.

Rest of world.


39


Product Contents

Reagent Bottle Rack

Rotor Adapter Holder

Rack for accommodating 6 x

30 ml reagent bottles on the

QIAcube worktable

Holder for 12 disposable rotor adapters; for use with the QIAcube

Products that can be ordered from BD and BD authorized distributors*

PAXgene Blood RNA Tubes (100) 100 Blood Collection Tubes

Blood Collection Set BD Vacutainer

®

Safety-Lok

™

6 Blood Collection Set: 21G,

0.75 inch needle, 12 inch tubing with luer adapter;

50 per box, 200 per case

BD Vacutainer One-Use

Holder

Case only for 13 mm and 16 mm diameter; 1000/case

BD Vacutainer Plus Serum

Tubes

13 x 75 mm 4.0 ml draw with

Red BD Hemogard closure and paper label; 100/box, 1000/case

Related products that can be ordered from QIAGEN

QuantiTect Reverse

Transcription Kit (50)

QuantiTect Reverse

Transcription Kit (200)

For 50 x 20 µl reversetranscription reactions: gDNA

Wipeout Buffer, Quantiscript

®

Reverse Transcriptase,

Quantiscript RT Buffer,

RT Primer Mix, RNase-Free Water

For 200 x 20 µl reversetranscription reactions: gDNA

Wipeout Buffer, Quantiscript

Reverse Transcriptase,

Quantiscript RT Buffer,

RT Primer Mix, RNase-Free Water

Cat. no.

990390

990392

762165

367281

364815

368975

205311

205313

* These blood collection accessories represent typical products that can be used with PAXgene Blood RNA

Tubes. To find out more about these accessories, including how to order, visit www.bd.com/vacutainer/products/venous.

40



Product Contents

QuantiTect Primer Assay (200)* For 200 x 50 µl reactions or

400 x 25 µl reactions: 10x

QuantiTect Primer Assay

(lyophilized) miScript Reverse Transcription

Kit (10)*

For 10 reactions: miScript Reverse

Transcriptase Mix, miScript RT

Buffer, RNase-Free Water miScript SYBR Green PCR Kit

(200)*

Human miScript Assay 96

Set V10.1 (20)*

Human miScript Assay 384

Set V10.1 (20)

For 200 reactions: QuantiTect

SYBR Green PCR Master Mix, miScript Universal Primer

714 miScript Primer Assays

(for 20 x 50 µl reactions) targeting human miRNAs in miRBase version

10.1; provided in 96-well plates

714 miScript Primer Assays

(for 20 x 50 µl reactions) targeting human miRNAs in miRBase version

10.1; provided in 384-well plates

Cat. no.

Varies

218060

218073

218421

218431

QuantiTect Kits and QuantiTect Primer Assays are intended for research use. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.

The miScript PCR System is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention, or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.

Visit www.qiagen.com/geneXpression to find out more about standardized solutions for gene expression analysis — from RNA preparation to real-time RT-PCR

* Larger kit sizes available; please inquire.


41

PreAnalytiX Worldwide

PreAnalytiX products are distributed by QIAGEN and BD companies

Australia • Orders 03-9840-9800 • Fax 03-9840-9888 • Technical 1-800-243-066

Austria • Orders 0800/28-10-10 • Fax 0800/28-10-19 • Technical 0800/28-10-11

Belgium • Orders 0800-79612 • Fax 0800-79611 • Technical 0800-79556

Brazil • Orders 0800-557779 • Fax 55-11-5079-4001 • Technical 0800-557779

Canada • Orders 800-572-9613 • Fax 800-713-5951 • Technical 800-DNA-PREP (800-362-7737)

China • Orders 0086 21 3865 3865 • Fax 0086 21 3865 3965 • Technical 800 988 0325, 800 988 0327

Denmark • Orders 80-885945 • Fax 80-885944 • Technical 80-885942

Finland • Orders 0800-914416 • Fax 0800-914415 • Technical 0800-914413

France • Orders 01-60-920-926 • Fax 01-60-920-925 • Technical 01-60-920-930 • Offers 01-60-920-928

Germany • Orders 02103-29-12000 • Fax 02103-29-22000 • Technical 02103-29-12400

Hong Kong • Orders 800 933 965 • Fax 800 930 439 • Technical 800 930 425

Ireland • Orders 1800-555-049 • Fax 1800-555-048 • Technical 1800-555-061

Italy • Orders 02-33430411 • Fax 02-33430426 • Technical 800-787980

Japan • Telephone 03-5547-0811 • Fax 03-5547-0818 • Technical 03-5547-0811

Korea (South) • Orders 1544 7145 • Fax 1544 7146 • Technical 1544 7145

Luxembourg • Orders 8002-2076 • Fax 8002-2073 • Technical 8002-2067

Mexico • Orders 01-800-7742-639 • Fax 01-800-1122-330 • Technical 01-800-7742-639

The Netherlands • Orders 0800-0229592 • Fax 0800-0229593 • Technical 0800-0229602

Norway • Orders 800-18859 • Fax 800-18817 • Technical 800-18712

Singapore • Orders 65-67775366 • Fax 65-67785177 • Technical 65-67775366

Spain • Orders 91-630-7050 • Fax 91-630-5145 • Technical 91-630-7050

Sweden • Orders 020-790282 • Fax 020-790582 • Technical 020-798328

Switzerland • Orders 055-254-22-11 • Fax 055-254-22-13 • Technical 055-254-22-12

UK • Orders 01293-422-911 • Fax 01293-422-922 • Technical 01293-422-999

USA • Orders 800-426-8157 • Fax 800-718-2056 • Technical 800-DNA-PREP (800-362-7737) www.qiagen.com

www.PreAnalytiX.com

42


Argentina, Uruguay and Paraguay • Orders 011 4551 7100

Australia • Orders 1 800 656 100 • Fax 1 800 656 110

Austria • Orders 43 1 7063660 • Fax 43 1 7063660-11

Belgium • Orders 32-53720408 • Fax 32-53720558

Brazil • Orders 0800 055 5654

Canada • Orders 800-268-5430 • Fax 800-565-0897

Denmark • Orders 45 43 43-45-66 • Fax 45 43-43-41-66

East Europe, Middle East & Africa (EMA) • Orders 971 4 3379525 • Fax: 971 4 03379551

Finland • Orders 358-9-88-70-780 • Fax 358-9-88-70-7817

France • Orders 33 476 683636 • Fax 33 476 683693

Germany • Orders 49 6221305553 • Fax 49 6221305377

Italy • Orders 390248204775 • Fax 390248204817 • Technical 390248240264

The Netherlands • Orders 31 20 6545 716 • Fax 31 20 5829 421

New Zealand • Orders 0800 572 468 • Fax 0800 572 469

Spain • Orders 34-91-848-8115 • Fax 34-91-848-8104

Sweden • Orders 46 8 775 51 00 • Fax 46 8 775 51 95

Switzerland • Orders 41 61 4852222 • Fax 41 61 4852200

UK • Orders 44 1-865-781-666 • Fax 44 1-865-781-528

USA • Orders 888-237-2762 • Fax 800-847-2220 • Technical 800-631-0174 www.bd.com

www.PreAnalytiX.com


43

PreAnalytiX Blood miRNA Kit, PAXgene Blood RNA Tubes Handbook

Assistant Bot

May 2009

PAXgene

For manual or automated purification of miRNA from whole blood

Important: To be used in conjunction with

PAXgene Blood RNA Tubes.

For Research Use Only. Not for use in diagnostic procedures.

Contents

Kit Contents

Shipping and Storage

Product Use Limitations

Product Warranty and Satisfaction Guarantee

Quality Control

Technical Assistance

Safety Information

Introduction

Principle and procedure

Equipment and Reagents to Be Supplied by User

Protocol: Manual Purification of Total RNA, Including miRNA, from Human Whole Blood Collected into

PAXgene Blood RNA Tubes

Protocol: Automated Purification of Total RNA,

Including miRNA, from Human Whole Blood Collected into PAXgene Blood RNA Tubes

Troubleshooting Guide

Appendix A: General Remarks on Handling RNA

Appendix B: Storage, Quantification, and

Determination of Quality of RNA

Appendix C: Using the QIAcube

Ordering Information

Ordering Information

Ordering Information

PreAnalytiX Worldwide

Key Features

Frequently Answers and Questions

What are the steps involved in using the PAXgene Blood miRNA Kit?

What type of RNA can be purified using this kit?

What are the storage conditions for the purified RNA?

Related manuals

PreAnalytiX Blood miRNA Kit, PAXgene Blood RNA Tubes Handbook

Assistant Bot

May 2009

PAXgene

For manual or automated purification of miRNA from whole blood

Important: To be used in conjunction with

PAXgene Blood RNA Tubes.

For Research Use Only. Not for use in diagnostic procedures.

Contents

Kit Contents

Shipping and Storage

Product Use Limitations

Product Warranty and Satisfaction Guarantee

Quality Control

Technical Assistance

Safety Information

Introduction

Principle and procedure

Equipment and Reagents to Be Supplied by User

Protocol: Manual Purification of Total RNA, Including miRNA, from Human Whole Blood Collected into

PAXgene Blood RNA Tubes

Protocol: Automated Purification of Total RNA,

Including miRNA, from Human Whole Blood Collected into PAXgene Blood RNA Tubes

Troubleshooting Guide

Appendix A: General Remarks on Handling RNA

Appendix B: Storage, Quantification, and

Determination of Quality of RNA

Appendix C: Using the QIAcube

Ordering Information

Ordering Information

Ordering Information

PreAnalytiX Worldwide

Key Features

Frequently Answers and Questions

What are the steps involved in using the PAXgene Blood miRNA Kit?

What type of RNA can be purified using this kit?

What are the storage conditions for the purified RNA?

Related manuals

Qiagen

QIAcube

Qiagen

QUAcube

Qiagen

QIAcube HT

Qiagen

QIAcube

PreAnalytiX

QIAsymphony PAXgene Blood RNA

PreAnalytiX

PAXgene Blood RNA Kit

Himedia

HTBM006

Qiagen

Q-Base

GALENVS

magenti

Thermo Fisher Scientific

RapidOut DNA Removal Kit

Qiagen

RNAprotect Cell Reagent

APPLICHEM

Decontamination solution, RNase-ExitusPlus