Protocol - Norgen Biotek Corp.

Protocol - Norgen Biotek Corp.
3430 Schmon Parkway
Thorold, ON, Canada L2V 4Y6
Phone: 866-667-4362  (905) 227-8848
Fax: (905) 227-1061
Email: [email protected]
Urine Cell-Free Circulating RNA Purification Kits
Product Insert
Product # 56900, 57000, 57100
Introduction
Recent evidence indicates that cell-free circulating RNA (cfc-RNA), including exosomal RNA in urine,
contains valuable information for the discovery of biomarkers that can help with early detection of certain
cancer types and for monitoring the disease status. The advantage for using urine as a source for cancer
biomarkers is that it can be acquired in large quantities without using invasive procedures. In addition,
repeated sampling from the same individual is applicable, which facilitates longitudinal studies. There are
many advantages favouring the use of urinary nucleic acids for cancer biomarker discovery over blood,
tissue samples or other bodily fluids, including: (1) urine is non-infectious for HIV and less infectious for
many other pathogens; (2) the profile of urinary nucleic acids is similar to that in plasma or serum; (3)
nucleic acid purification from urine is technically much easier because of its low protein concentration (1000fold lower than blood).
Norgen’s Urine Cell-Free Circulating RNA Purification Kits provide a fast, reliable reproducible and simple
procedure for isolating circulating RNA and exosomal RNA from various urine inputs ranging from 250 µL
and up to 30 mL, with various kit formats addressing different urine input volumes. Purification is based on
spin column chromatography that uses Norgen’s proprietary resin separation matrix. The kit is designed to
isolate all sizes of circulating RNA, including microRNA, as well as all sizes of exosomal RNA. Norgen’s
Urine Cell-Free Circulating RNA Purification Kits provides a clear advantage over other available kits in that
they do not require phenol/chloroform or any protease treatments. Moreover, the kit allows the user to elute
into a flexible elution volume ranging from 50 µL to 100 µL. The purified RNA is of the highest integrity, and
can be used in a number of downstream applications including real time PCR, reverse transcription PCR,
Northern blotting, RNase protection and primer extension, and expression array assays.
Kit Descriptions and Components
Number of Preps
Mini Kit
(Cat.# 56900)
50 preps
Midi Kit
(Cat.# 57000)
20 preps
Binding Solution K
25 mL
75 mL
Lysis Buffer A
30 mL
20 mL
Maxi Kit
(Cat.# 57100)
10 preps
1 x 75 mL
1 x 25 mL
20 mL
Wash Solution A
18 mL
18 mL
18 mL
Elution Solution A
6 mL
6 mL
6 mL
Mini Spin Columns
50
20
10
Midi Spin Column
20
Maxi Spin Column
10
Collection Tubes
50
20
10
Elution tubes (1.7 mL)
50
20
10
Product Insert
1
1
1
These kits are suitable for the isolation of cfc-RNA from fresh urine samples, frozen urine samples or urine
samples collected on any urine preservative.
It has been noticed that urine samples stored at -70°C, -20°C or at 4°C will develop some precipitation due
to the aggregation of some of the highly abundant proteins in urine. Eliminating these precipitates using
centrifugation or filtration may cause the loss of some of the cfc protein-bound RNA. Furthermore, these
precipitates may affect the quality of the purified nucleic acid. We recommend the use of Norgen’s Urine
Preservative when collecting urine samples. Norgen’s Urine Preservative is designed for the preservation of
nucleic acids and proteins in fresh urine samples at ambient temperatures, therefore no protein precipitation
will occur and the purified nucleic acids will be of a higher quality. The components of the Urine Preservative
allow samples to be stored for over 2 years at room temperature with no detected degradation of urine DNA,
RNA or proteins. Norgen’s Urine Preservative is available as a liquid format in Norgen’s Urine Preservative
Single Dose Ampules, as well as in a dried format in Norgen’s Urine Collection and Preservation Tubes
1
Kits Specifications
Sample Type
Mini Kit
Cat.# 56900
Fresh or frozen urine
Midi Kit
Cat.# 57000
Fresh or frozen urine
Maxi Kit
Cat.# 57100
Fresh or frozen urine
Sample volume Range
250 µL to 2 mL
2 to 10 mL
10 to 30 mL
Minimum Elution Volume
50 µL
50 µL
50 µL
Maximum Elution Volume
100 µL
100 µL
100 µL
Time to Complete 10 Purifications
25 - 30 minutes
40 - 45 minutes
45 - 50 minutes
¥
Size of RNA Purified
All sizes, including miRNA and small RNA (< 200 nt)
Average Yields ¥
Variable depending on specimen
Please check page 6 for Average Urine Yields and Common RNA Quantification Methods
Customer-Supplied Reagents and Equipments

Benchtop microcentrifuge

Swinging bucket centrifuges

Vortexer

Micropipettors

96 – 100% ethanol

100% Isopropanol

β - Mercaptoethanol
Storage Conditions and Product Stability
o
All buffers should be kept tightly sealed and stored at room temperature (15-25 C) for up to 2 year without
o
showing any reduction in performance. It is recommended to warm Lysis Buffer A for 20 minutes at 60 C if
any salt precipitation is observed.
Quality Control
In accordance with Norgen’s Quality Management System, each lot of Norgen’s Urine Cell-Free Circulating
RNA Purification Kits are tested against predetermined specifications to ensure consistent product quality.
Product Use Limitations
Norgen’s Urine Cell-Free Circulating RNA Purification Kits are designed for research purposes only. It is not
intended for human or diagnostic use.
Product Warranty and Satisfaction Guarantee
NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in
our product manual. The customer must determine the suitability of the product for its particular use.
Safety Information
Ensure that a suitable lab coat, disposable gloves and protective goggles are worn when working with
chemicals. For more information, please consult the appropriate Material Safety Data Sheets (MSDSs).
These are available as convenient PDF files online at www.norgenbiotek.com.
Binding Solution K and Lysis Buffer A contain guanidium salt, and should be handled with care.
Guanidium salt forms highly reactive compounds when combined with bleach, thus care must be taken to
properly dispose of any of these solutions.
If liquid containing these solutions 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.
CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.
2
Important Notes
Working with RNA
RNases are very stable and robust enzymes that degrade RNA. Autoclaving solutions and glassware is not
always sufficient to actively remove these enzymes. The first step when preparing to work with RNA is to
create an RNase-free environment. The following precautions are recommended as your best defence
against these enzymes.

The RNA area should be located away from microbiological work stations

Clean, disposable gloves should be worn at all times when handling reagents, samples, pipettes,
disposable tubes, etc. It is recommended that gloves are changed frequently to avoid
contamination

There should be designated solutions, tips, tubes, lab coats, pipettes, etc. for RNA only

All RNA solutions should be prepared using at least 0.05% DEPC-treated autoclaved water or
molecular biology grade nuclease-free water

Clean all surfaces with commercially available RNase decontamination solutions

When working with purified RNA, ensure that they remain on ice during downstream applications
Notes Prior to Use
 All centrifugation steps are performed at room temperature.
 Ensure that centrifuge tubes used are capable of withstanding the centrifugal forces required.
 The provided spin columns are optimized to be used with a benchtop centrifuges and not to be
used on a vacuum apparatus
 Most standard benchtop microcentrifuges will accommodate Norgen's Mini Spin Columns.
 Most standard swinging bucket centrifuges will accommodate Norgen's Midi and Maxi Spin
Columns. Do not use a fixed-angle rotor
 Norgen's Midi and Maxi Spin Columns are centrifuged in 15 mL and 50 mL centrifuge tubes,
respectively.
 Centrifuging Norgen's Spin columns at a speed higher than recommended may affect RNA yield.
 Centrifuging Norgen's Spin columns at a speed lower than recommended will not affect RNA yield.
However, centrifugation at a lower speed may require longer time for the solutions to pass through
the spin column
 When placing Norgen's Midi and Maxi Spin Columns into the swinging bucket centrifuge make sure
that lids of the tubes are not tightly closed. Tightly closed lids may cause back pressure which may
cause column clogging or disintegration.
 Ensure that all solutions are at room temperature prior to use.
o
 It is highly recommended to warm up Lysis Buffer A at 60 C for 20 minutes and mix well until the
solutions become clear again if precipitates are present.
 Prepare a working concentration of the Wash Solution A by adding 42 mL of 96 - 100% ethanol
(provided by the user) to the supplied bottle containing 18mL of concentrated Wash Solution A.
This will give a final volume of 60 mL. The label on the bottle has a box that may be checked to
indicate that the ethanol has been added.
 The use of β-mercaptoethanol in lysis is highly recommended to isolate RNA for sensitive
downstream applications. Add 10 µL of β-mercaptoethanol (provided by the user) to each 1 mL of
Lysis Buffer A.
 If any of the solutions do not go through the Spin Columns within the specified
centrifugation time, spin for an additional 1-2 minutes until the solution completely passes
through the Column. Do NOT exceed the centrifugation speed as this may affect RNA yield.
Preparation of Cell-free Urine Sample
1.
2.
3.

Collect and transfer 15-50 mL of the urine into a conical tube and centrifuge at 200 x g (~1,000
RPM) for 10 minutes to remove urine exfoliated cells and debris. Decant cell-free urine into new
15-50 mL conical tube.
Centrifuge the cell-free urine at 1,800 x g (~3,000 RPM) for 10 minutes to remove any residual
debris or bacterial cells.
Transfer cell-free urine into a fresh 15-50 mL conical tube.
Cell-Free Urine is now ready for the purification of cell-free RNA
Please check the product # and proceed to the appropriate section for
your Urine cfc-RNA Purification
3
Section 1: Urine Cell-Free Circulating RNA Purification Mini Kit (Cat. #56900)
Note: The procedure outlined below is for 2 mL inputs of urine. If processing a sample volume lower
than 2 mL urine, simply bring the volume of your samples up to 2 mL using Nuclease-free water and
proceed as outlined below.
1.
Place 2 mL of cell-free urine in a 15 mL tube (provided by the user). Add 400 µL Binding Solution K
and mix well by vortexing for 10 seconds.
2. Transfer 800 µL of the mixture from Step 1 into a Mini Spin column assembled with one of the provided
collection tubes. Centrifuge for 2 minutes at 3,300 x g (~7,000 RPM). Discard the flowthrough and
reassemble the spin column with its collection tube.
3. Repeat Step 2 two more times to transfer the remaining mixture into the Mini Spin column.
4. Apply 500 µL of Lysis Buffer A to the column and centrifuge for 1 minute at 3,300 x g (~7,000 RPM).
Do Not Discard the flowthrough.
5. Reload the flowthrough from Step 4 back to the column and let stand at room temperature for 10
minutes. Centrifuge for 1 minute at 3,300 x g (~7,000 RPM). Do Not Discard the flowthrough.
6. To the flowthrough from Step 5 add 250 µL 100% Isopropanol and mix well by pipetting.
7. Transfer the entire mixture from Step 6 back to the Mini Spin column assembled with one of the
provided collection tubes. Centrifuge for 2 minutes at 3,300 x g (~7,000 RPM). Discard the
flowthrough and reassemble the spin column with its collection tube.
8. Apply 600 µL of Wash Solution A to the column and centrifuge for 1 minute at 3,300 x g (~7,000
RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
9. Repeat Step 8 one more time, for a total of two washes.
10. Spin the column, empty, for 2 minutes at 14,000 x g (~14,000 RPM). Discard the collection tube.
11. Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 50 µL of Elution Solution A to the
column and let stand at room temperature for 2 minutes. Centrifuge for 1 minute at 200 x g (~2,000
RPM), followed by 2 minutes at 5,200 x g (~8,000 RPM).
12. For maximum recovery, transfer the eluted buffer back to the column and let stand at room temperature
for 2 minutes. Centrifuge for 1 minute at 400 x g (~2,000 RPM), followed by 2 minutes at 5,800 x g
(~8,000 RPM).

Urine cfc-RNA is ready for the downstream application of your choice. For an
explanation of expected yields and recommendations for quantification of the
RNA, please refer to Appendix A
Section 2: Urine Cell-Free Circulating RNA Purification Midi Kit (Cat. #57000)
Note: The procedure outlined below is for 10 mL inputs of urine. If processing a sample volume
lower than 10 mL urine, simply bring the volume of your samples up to 10 mL using Nuclease-free
water and proceed as outlined below.
1.
2.
3.
4.
5.
6.
7.
Place 10 mL of cell-free urine in a 50 mL tube (provided by the user). Add 3 mL Binding Solution K
and mix well by vortexing for 10 seconds.
Transfer 4.5 mL of the mixture from Step 1 into a Midi Spin column assembled with one of the provided
collection tubes. Centrifuge for 3 minutes at 1,000 x g (~2,200 RPM). Discard the flowthrough and
reassemble the spin column with its collection tube.
Repeat Step 2 two more times to transfer the remaining mixture into the Midi Spin column.
Apply 400 µL of Lysis Buffer A to the column and centrifuge for 3 minutes at 500 x g (~1,600 RPM).
Do Not Discard the flowthrough.
Apply an additional 400 µL of Lysis Buffer A to the column and centrifuge for 3 minutes at 500 x g
(~1,600 RPM). Do Not Discard the flowthrough.
Reload the 800 µL flowthrough from Step 5 back to the column and let stand at room temperature for
20 minutes. Centrifuge for 3 minutes at 1,000 x g (~2,200 RPM). Do Not Discard the flowthrough.
To the flowthrough from Step 6 add 400 µL 100% Isopropanol and mix well by pipetting.
4
8.
9.
10.
11.
12.
13.
Transfer 600 µL of the mixture from Step 7 to a Mini Spin column assembled with one of the provided
collection tubes. Centrifuge for 2 minutes at 3,300 x g (~7,000 RPM). Discard the flowthrough and
reassemble the spin column with its collection tube.
Repeat Step 8 one more time to transfer the remaining mixture into the Mini Spin column.
Apply 600 µL of Wash Solution A to the column and centrifuge for 1 minute at 3,300 x g (~7,000
RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
Repeat Step 10 one more time, for a total of two washes.
Spin the column, empty, for 2 minutes at 14,000 x g (~14,000 RPM). Discard the collection tube.
Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 50 µL of Elution Solution A to the
column and let stand at room temperature for 2 minutes. Centrifuge for 1 minute at 200 x g (~2,000
RPM), followed by 2 minutes at 5,200 x g (~8,000 RPM).
14. For maximum recovery, transfer the eluted buffer back to the column and let stand at room temperature
for 2 minutes. Centrifuge for 1 minute at 400 x g (~2,000 RPM), followed by 2 minutes at 5,800 x g
(~8,000 RPM).

Urine cfc-RNA is ready for the downstream application of your choice. For an
explanation of expected yields and recommendations for quantification of the
RNA, please refer to Appendix A
Section 3: Urine Cell-Free Circulating RNA Purification Maxi Kit (Cat. #57100)
Note: The procedure outlined below is for 30 mL inputs of urine. If processing a sample volume
lower than 30 mL urine, simply bring the volume of your samples up to 30 mL using Nuclease-free
water and proceed as outlined below.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Place 30 mL of cell-free urine in a 50 mL tube (provided by the user). Add 9 mL Binding Solution K
and mix well by vortexing for 10 seconds.
Transfer 13 mL of the mixture from Step 1 into a Maxi Spin column assembled with one of the provided
collection tubes. Centrifuge for 3 minutes at 1,000 x g (~2,200 RPM). Discard the flowthrough and
reassemble the spin column with its collection tube.
Repeat Step 2 two more times to transfer the remaining mixture into the Maxi Spin column.
Apply 600 µL of Lysis Buffer A to the column and centrifuge for 3 minutes at 500 x g (~1,600 RPM).
Do Not Discard the flowthrough.
Apply an additional 600 µL of Lysis Buffer A to the column and centrifuge for 3 minutes at 500 x g
(~1,600 RPM). Do Not Discard the flowthrough.
Reload the 1.2 mL flowthrough from Step 5 back to the column and let stand at room temperature for
30 minutes. Centrifuge for 3 minutes at 1,000 x g (~2,200 RPM). Do Not Discard the flowthrough.
To the flowthrough from Step 6 add 600 µL 100% Isopropanol and mix well by pipetting.
Transfer 600 µL of the mixture from Step 7 to a Mini Spin column assembled with one of the provided
collection tubes. Centrifuge for 2 minutes at 3,300 x g (~7,000 RPM). Discard the flowthrough and
reassemble the spin column with its collection tube.
Repeat Step 8 two more time to transfer the remaining mixture into the Mini Spin column.
Apply 600 µL of Wash Solution A to the column and centrifuge for 1 minute at 3,300 x g (~7,000
RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
Repeat Step 10 one more time, for a total of two washes.
Spin the column, empty, for 2 minutes at 14,000 x g (~14,000 RPM). Discard the collection tube.
Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 50 µL of Elution Solution A to the
column and let stand at room temperature for 2 minutes. Centrifuge for 1 minute at 200 x g (~2,000
RPM), followed by 2 minutes at 5,200 x g (~8,000 RPM).
14. For maximum recovery, transfer the eluted buffer back to the column and let stand at room temperature
for 2 minutes. Centrifuge for 1 minute at 400 x g (~2,000 RPM), followed by 2 minutes at 5,800 x g
(~8,000 RPM).

Urine cfc-RNA is ready for the downstream application of your choice. For an
explanation of expected yields and recommendations for quantification of the
RNA, please refer to Appendix A
5
Appendix A
Cell-Free Circulating RNA Yield
Urine cfc-RNA, like RNA in other cell-free bodily fluids, is normally found in very low amounts (1 - 100
pg/µL), therefore measuring cell-free RNA concentration using common quantification methods is very
difficult and challenging. Typical yields of urine cfc-RNA vary significantly from sample to sample. Variability
is also observed between samples collected from the same donor at different times during the day and
therefore there is no absolute yield for RNA purified from bodily fluids including urine. Cell-free circulating
RNA yield varies depending on a number of factors including age, sex, diet, exercise and most importantly
the health status of the donor.
Below is a list of the most common RNA quantification methods, as well as the limit of detection for each of
these methods. Unfortunately, none of these methods can be used reliably for measuring the
concentration of RNA purified from urine unless large urine volumes have been processed. This
would only be applicable if urine contains the maximum amount of RNA that can fit within the specification
range of these quantification tools. It should be noted that the specifications outlined below are based on
measuring a pure RNA, which will not be the case for the RNA purified from urine. Urine RNA is short
fragmented RNA which is usually present in less than 1000 bp. Purified urine RNA usually contains traces of
proteins which will interfere with most quantification methods, leading to the overestimation of the purified
RNA concentration. Therefore purified RNA contaminated with more proteins will be presented at a higher
concentration as compared to RNA purified with less protein contaminants, which in this case will depend on
the method used for urine RNA purification. The only reliable method that can assess the quality and
the relative quantity of the purified urine RNA is RT-qPCR amplification of a standard RNA using a
small RNA amplicon such as the 5S rRNA housekeeping gene.
Common RNA Quantification Methods
1) Bioanalyzer RNA Quantification kits
RNA 6000 Nano Kit
RNA 6000 Pico Kit
Small RNA kit
Total RNA
mRNA
Total RNA
mRNA
Total RNA
Quantitative range
25 - 500 ng/µL
25 - 250 ng/µL
5 - 500 ng/µL
5 - 250 ng/µL
---250 - 5000
pg/µL
50-2000 pg/µL
Qualititative range
---50 - 5000
pg/µL
Quantitation
accuracy
20% CV
20% CV
30% CV
----
------
50-2000 pg/µL
2) NanoDrop 2000

Detection Limit: 2 ng/µl (dsDNA)
3) Quant-iT™ RiboGreen® RNA Assay Kit

Quantitation Range: 1-200 ng
4) qPCR Standard Curve (generated by Norgen)
30 fg
300 fg
3 pg
10 pg
30 pg
100 pg
300 pg
6
Frequently Asked Questions
1. What If a variable speed centrifuge is not available?
 A fixed speed centrifuge can be used, however reduced yields may be observed.
2. At what temperature should I centrifuge my samples?
 All centrifugation steps are performed at room temperature. Centrifugation at 4C will not adversely
affect kit performance.
3. What if I added more or less of the specified reagents’ volume?
 Adding more or less than the specified volumes may reduce both the quality and the quantity of the
purified RNA. Eluting your RNA in high volumes will increase the yield but will lower the concentration.
Eluting in small volumes will increase the concentration but will lower the overall yield.
4. What if I forgot to do a dry spin before my final elution step?
 Your purified RNA will be contaminated with the Wash Solution A. This may reduce the quality of your
purified RNA and will interfere with your downstream applications.
5. Can I perform a second elution?
nd
st
 Yes, but it is recommended that the 2 elution be in a smaller volume (50% of 1 Elution). It is also
nd
st
recommended to perform the 2 elution into a separate elution tube to avoid diluting the 1 elution.
6. Why do my samples show low RNA yield?
 Urine samples contain very little RNA. This varies from individual to individual based on numerous
variables. In order to increase the yield, the amount of urine input could be increased.
7. Why do the A260:280 ratio of the purified RNA is lower than 2.0?
 Most of the Free-Circulating Urine RNA is short RNA fragments. The A260:280 ratio is normally
between 1 – 1.6. This low A260:280 ratio will not affect any downstream application.
8. Why does my isolated RNA not perform well in downstream applications?
 If a different Elution Buffer was used other than the one provided in the kit, the buffer should be
checked for any components that may interfere with the application. Common components that are
known to interfere are high salts (including EDTA), detergents and other denaturants. Check the
compatibility of your elution buffer with the intended use.
Technical Assistance
NORGEN’s Technical Service Department is staffed by experienced scientists with extensive practical and
theoretical expertise in sample and assay technologies and the use of NORGEN products. If you have any
questions or experience any difficulties regarding Norgen’s Urine Cell-Free Circulating RNA Purification Kits
or NORGEN products in general, please do not hesitate to contact us.
NORGEN customers are a valuable 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 NORGEN. 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 contact our Technical Support Team between the
hours of 8:30 and 5:30 (Eastern Standard Time) at (905) 227-8848 or Toll Free at 1-866-667-4362. or call
one of the NORGEN local distributors (www.norgenbiotek.com) or through email at
[email protected]
3430 Schmon Parkway, Thorold, ON Canada L2V 4Y6
Phone: (905) 227-8848
Fax: (905) 227-1061
Toll Free in North America: 1-866-667-4362
©2015 Norgen Biotek Corp.
PI56900-3
7
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement