User Manual-ENZ-42421-100 - Rev 1.1.0 April 2010.pub

Enabling Discovery in Life Science®
www.enzolifesciences.com
Enabling Discovery in Life Science®
100-Reaction Single-Round RNA Amplification
and Biotin Labeling System
Instruction Manual
Cat. No. ENZ-42421-100
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For research use only.
Rev. 1.1.0 April 2010
Problem
Potential Cause
Suggestion
Incorrect incubation
temperatures
Check the accuracy of the temperatures of your incubators and thermal
cycler.
Reaction tube condensation
Briefly centrifuge the reaction tube(s).
Mix gently and move the reaction tube
(s) to an incubator where condensation
does not occur.
RNA sample vacuum
centrifuged to dryness
Monitor sample volume during vacuum
centrifugation to avoid drying to completion. RNA is difficult to resuspend if
dried completely.
Low yields with both
experimental samples and control RNA
Inadequate elution of
(continuation..)
cDNA
Heat elution buffer to 70°C. Proper
maintenance of temperature at this
step is critical for maximal elution of
cDNA from the column filter.
Inadequate elution of
aRNA
Elute nucleic acids from column filter
purification systems with enough elution solution to completely saturate the
filter. Dispense the elution buffer in the
center of the column bed.
Incorrect spectrophotometer readings
Check the accuracy of your spectrophotometer. Alternatively, use another
method for quantitation of aRNA such
as staining with a sensitive RNA dye.
Notice to Purchaser
This product is manufactured and sold by ENZO LIFE SCIENCES, INC. for research use
only by the end-user in the research market and is not intended for diagnostic or
therapeutic use. Purchase does not include any right or license to use, develop or
otherwise exploit this product commercially. Any commercial use, development or
exploitation of this product or development using this product without the express prior
written authorization of ENZO LIFE SCIENCES, INC. is strictly prohibited.
Limited Warranty
This product is offered under a limited warranty. The product is guaranteed to meet
appropriate specifications described in the package insert at the time of shipment. Enzo
Life Sciences’ sole obligation is to replace the product to the extent of the purchase price.
All claims must be made to Enzo Life Sciences, Inc. within five (5) days of receipt of
order.
Average size of control aRNA is greater
than 1 kb but average size of experimental aRNA is less
than 0.5 kb.
Prepare fresh samples following the
guidelines in Appendix A to prevent
RNase contamination. Use nucleasefree plasticware and decontaminate
pipettes and work surfaces with commercially available RNase decontamination solutions.
RNA is partially degraded.
Trademarks and Patents
Enzo, is a trademark of Enzo Life Sciences, Inc. Several of Enzo’s products and product
applications are covered by US and foreign patents and patents pending.
12
VIII. Troubleshooting Guide
Contents
Control RNA Reaction
High quality Control RNA (100 ng/L, Cat. No. ENZ-42421-CR) can be
used to help evaluate kit performance for troubleshooting and as an aid
to first-time users. Parallel reactions with sample and control RNA (5 L,
500 ng) can be performed. Typical yield of purified aRNA generated
from a 16 hr in vitro transcription reaction with 500 ng of input control
RNA in the first strand cDNA synthesis reaction should be ≥ 30 g. In
addition, 100 ng of the aRNA generated from control RNA can be analyzed on a bioanalyzer and results compared to the profile in Figure 1
(Appendix C).
Problem
Low aRNA yields with
experimental samples
Potential Cause
Suggestion
Organic, protein or salt
contaminants in the input
RNA
Perform parallel reaction with control RNA
(ENZ-42406-CR, sold separately). Use 5 uL
(500 ng) in the first strand synthesis reaction.
Typical yield from this input is 30 ug or more.
If control RNA amplifies to this level but not
your experimental samples, repurify your
starting material. Use extra caution not to
carry over organic organic solvents or denaturants during purification.
Increase the total RNA input if the experimental RNA sample is expected to have low
mRNA content.
Input RNA does not contain
poly(A) tracts
RNA amplification with this kit requires the
use of oligo dT primers. Do not substitute
random or gene-specific primers with this kit.
Degraded input RNA
Prepare fresh sample following the guidelines in Appendix A to prevent RNase contamination. Use nuclease-free plasticware
and decontaminate pipettes and work surfaces with commercially available RNase
decontamination solutions.
Inadequate thawing and
mixing of reagents
Ensure reagents are completely
thawed with no solids remaining.
Gently mix and centrifuge reagents
briefly.
Nuclease contamination
Prepare fresh samples following the
guidelines in Appendix A to prevent
RNase contamination. Use nucleasefree plasticware and decontaminate
pipettes and work surfaces with commercially available RNase decontamination solutions.
Continued…….
11
II. Procedural Overview ................................................ 1
III. Reagents Provided and Storage.............................. 2
IV. Additional Materials Required ................................. 2
V. Methods and Procedures ......................................... 3
Low mRNA content of experimental sample
Low yields with both
experimental samples and control RNA
I. Introduction ............................................................... 1
A. FIRST-STRAND SYNTHESIS ............................................... 4
B. SECOND-STRAND SYNTHESIS ........................................... 5
C. CDNA PURIFICATION ........................................................ 5
D. RNA TRANSCRIPT LABELING............................................. 6
E. aRNA PURIFICATION ........................................................ 6
VI. Appendices ............................................................... 7
A. RECOMMENDATIONS FOR RNASE-FREE TECHNIQUE........... 7
B. RNA PREPARATION GUIDELINES ....................................... 7
C. cDNA AND aRNA PURIFICATION GUIDELINES .................... 8
D. ANALYSIS OF BIOTIN-LABELED TRANSCRIPT..................... 10
E. aRNA FRAGMENTATION ................................................. 10
VII. References .............................................................. 10
VIII. Troubleshooting Guide ......................................... 11
D. ANALYSIS OF BIOTIN-LABELED TRANSCRIPT
I. Introduction
The 100-Reaction Single-Round RNA Amplification and Biotin Labeling System provides an optimized protocol and reagents for the production of biotin-labeled antisense RNA (aRNA) from total cellular RNA samples (0.25 g – 5 g) in less than 24 hours for array analysis. The 100Reaction Single-Round RNA Amplification and Biotin Labeling System
has been optimized for superior performance across several automated
liquid handling platforms. The result is a comprehensive system that reduces variability while subsequently improving reproducibility, data quality
and throughput for automated genomic environments.
The complete system is composed of reagents for cDNA synthesis and in
vitro transcription labeling and does not provide materials required for
purification.
The RNA yield can be estimated by absorbance detection at 260 nm
and 280 nm using a UV/Vis spectrophotometer. If using a NanoDrop Spectrophotometer, assay 2 µL of the aRNA sample directly.
If analyzing by Agilent 2100 Bioanalyzer or similar method, the distribution of biotin-labeled aRNA should range from 200-6000 nt with a
broad peak between 1 kb and 2 kb (Figure 1).
If a bioanalyzer is not available, the quality of the amplified aRNA
can be assessed by gel electrophoresis. For high quality total RNA,
the resulting aRNA appear as a smear of RNAs ranging from 200 nt
to 5000 nt with and average size of approximately 1200 nt.
II. Procedural Overview
First Strand cDNA Synthesis
 Add 1 L T7 Oligo(dT) Primer to 0.25 - 5 g total
RNA.
 Bring total volume to 13 L with nuclease-free water.
 Incubate at 70˚C for 10 minutes and move to 42°C.
 Add 7 L of First Strand Master Mix.
 Incubate at 42˚C for 2 hours.
Second Strand cDNA Synthesis
 Add 130 L Second Strand Master Mix.
 Incubate at 16˚C for 2 hours.
Figure 1. Analysis of biotin-labeled aRNA using the Single-Round RNA Amplification
and Labeling System. After purification, 400 ng of synthesized aRNA was evaluated using
the RNA Nano/Pico LabChip kit on an Agilent 2100 BioAnalyzer.
cDNA Purification
Note: This kit does not include consumables or reagents
required for purification. Please refer to the Appendix for
suggestions and guidelines.
In Vitro Transcription and Biotin Labeling
 Add 27 L IVT Master Mix.
 Incubate at 37˚C for 6 hours for input RNA > 2 g.
 Incubate at 37˚C for 16 hours for input RNA
0.25-2 g.
E. aRNA FRAGMENTATION
For proper hybridization to microarrays, labeled aRNAs should be
reduced in size. Follow the guidelines of your microarray manufacturer regarding aRNA fragmentation. In general, any RNA hydrolysis method that generates RNA fragments in the 75 – 200 nucleotide
size range is acceptable
VII. References
1. Van Gelder et al. PNAS 87(1990)1663-7. Van Gelder et al.
PNAS 87(1990)
1
10
II. aRNA
III. Reagents Provided and Storage
1.
Equilibrate the RNAClean reagents to room temperature and
then resuspend the magnetic beads during the last 30 min of
the IVT reaction.
2.
Transfer the samples from the IVT reaction into a 96-well Ubottom plate.
3.
Add 108 µL of magnetic beads to each sample and incubate
for 5 minutes at room temperature.
4.
5.
6.
7.
8.
Table 1. Kit Components
Min. Vol. Supplied
Vial ID
dNTP Mix
400 µL
dN
Promoter Primer
100 µL
P
First Strand Buffer
200 µL
FB
Gently remove the supernatant while keeping the plate on
the magnetic stand.
DTT
800 µL
D
Reverse Transcriptase
100 µL
RT
Remove the plate from the magnetic stand and wash the
beads with 200 µL of 70% ethanol (freshly prepared) and
incubate for 30 seconds at room temperature.
RNase Inhibitor
100 µL
I
DNA Polymerase
500 µL
DP
RNase H
100 µL
RH
Separate by placing the 96-well plate on the magnetic stand
for approximately 5 minutes or until the mixture is clear.
Second Strand Buffer
1.5 mL
SB
IVT Reaction Buffer
600 µL
RB
Aspirate and discard the supernatant and repeat steps 6 -7.
Aspirate trace amounts of ethanol without disturbing the
beads.
10X Biotin-Labeled Ribonucleotide
600 µL
B
Enhancer Cocktail
600 µL
EC
T7 RNA Polymerase
300 µL
T7
Nuclease-free Water
13 mL
W
Separate by placing the 96-well plate on the magnetic stand
for 10 minutes or until the mixture is clear.
9.
Remove the plate from the magnetic stand and allow the
beads to air dry for 10 minutes.
10.
Add 62 µL of RNase-free water (25˚C) to each well, seal and
vortex or shake for 1 minute.
11.
Store all kit components at –20°C in a non-frost-free freezer. Water can
be left at room temperature after first thawing.
Separate the beads by applying the magnet for 10 minutes
or until clear.
12.
Collect and transfer 60 µL of aRNA solution to a 96-well
PCR plate.
13.
Analyze the RNA as required, or store at -20˚C overnight or
at -80˚C for long term storage.
Reagents
Table 2. Related Product (Available Separately)
Reagent
Control RNA (100 µg/mL)
Supplied
Cat. No.
10 µL
ENZ-42406-CR
IV. Additional Materials Required
Equipment






9
Thermal cycler with heated lid or heating blocks with heated lid
Cold block (4°C).
Microcentrifuge
Orbital shaker for 96-well plates (optional)
UV Spectrophotometer (optional)
Bioanalyzer (Agilent, optional)
2
C. cDNA AND aRNA PURIFICATION GUIDELINES
Materials





Isolated and purified RNA
0.2 ml, 0.6 ml and 1.5 ml microcentrifuge tubes
Sterile, aerosol barrier, nuclease-free pipette tips
RNase decontamination solution
Molecular Biology Grade Ethanol (100%)
There are several commercially available column filter-based purification systems for the purification of the aRNA. To avoid precipitation of reagents or sample, ensure that all solutions are at room temperature and thoroughly mixed before use. All excess wash solution
must be removed from the column filter prior to aRNA elution. .
The 100-Reaction Single-Round RNA Amplification and Labeling
System is compatible with the Agencourt® BioScience RNAClean™
purification system and is recommended using the following protocols.
V. Methods and Procedures
NOTE: PLEASE READ THE ENTIRE PROCEDURE BEFORE STARTING. Upon
thawing of solutions, gently hand-mix or vortex the reagents prior to use to
ensure a homogenous solution.
The entire process involves four major steps that are accomplished by
using the 100-Reaction Single-Round Amplification and Biotin Labeling
System with recommended purification and analysis procedures.
With the exception of all purifications, the entire procedure may be
performed in a thermal cycler (see Table 3, below). To avoid condensation, set the thermal cycler lid temperature to 5°C above the reaction
temperature. Alternatively, heating blocks with heated lids, water baths,
or hybridization ovens set at the prescribed temperatures can be used.
For the sake of consistency of common reaction components, we recommend making master mixes for each step of the procedure when performing more than one reaction. The reaction tables show exact reactant volumes per reaction. While making a master mix, Include 10% extra volume
of each common reaction component in the master mix to make up for
losses in distributing into each reaction vial.
Table 3. Thermal Cycler Amplification Program
Process
°C
Time
Denaturation
70°
42°
42°
10 minutes
3 minutes
Pause
Reverse Transcription
42°
4°
120 minutes
Pause
Second Strand Synthesis
16°
4°
120 minutes
Pause
In Vitro Transcription
37°
4°
16 hours (see step D-5)
Pause
3
I.
cDNA
1. Equilibrate the RNAClean reagents to room temperature and
then resuspend the magnetic beads during the last 30 min of
second strand cDNA synthesis.
2. Add 135 µL of RNAClean™ to each well of a 96-well
U-bottom plate. Add 75 µL of each second strand reaction
to the plate containing beads and incubate for 5 minutes at
room temperature.
3.
Separate by placing the 96-well plate on the magnetic stand
for 10 minutes or until the mixture is clear.
4.
Gently remove the supernatant while keeping the plate on
the magnetic stand.
5.
Repeat Steps 1-4 with the remaining second strand reaction
volume.
6.
Remove the plate from the magnetic stand and wash the
beads with 200 µL of 70% ethanol (freshly prepared) and
incubate for 30 seconds at room temperature.
7.
Separate by placing the 96-well plate on the magnetic stand
for approximately 5 minutes or until the mixture is clear.
8.
Aspirate and discard the supernatant and repeat steps 6 -7.
Aspirate trace amounts of ethanol without disturbing the
beads.
9.
Remove the plate from the magnetic stand and allow the
beads to air dry for 10 minutes.
10.
Add 36 µL of RNase-free water (25˚C) to each well and gently shake on orbital shaker for 2 minutes.
11.
Separate the beads by applying the magnet for 10 minutes
or until clear.
12.
Collect and transfer 33 µL of cDNA solution to a 96-well
PCR plate.
13.
Purified cDNA can be stored at -20˚C for up to 3 days before
use.
8
A. FIRST-STAND SYNTHESIS
VI. Appendices
A. RECOMMENDATIONS FOR RNASE-FREE TECHNIQUE
Avoid all sources of RNase contamination as generation of high
quality labeled aRNA product requires that full length RNA be maintained from sample preparation to cDNA synthesis and transcription.

Wear disposable powder-free gloves and change frequently
throughout.

Avoid RNase containing surfaces.

Use only the reagents supplied in this kit. Other reagents may not
be compatible.

Use only sterile, DNase and RNase-free pipet tips, microcentrifuge
tubes and other plasticware.

Clean and decontaminate pipettes and work surfaces with commercially available RNase decontaminating solutions or wipes.
B. RNA PREPARATION GUIDELINES

RNA purity and integrity affect both the quantity and quality of the
resulting cDNA .

Starting with total cellular RNA minimizes loss of low abundance
transcripts during mRNA isolation.


Most commercially available RNA isolation reagents are compatible
with the 100-Reaction Single-Round RNA Amplification and Labeling System.
Total RNA samples should be in water or buffer, free of protein,
DNA, cellular material, organic solvents, salts and other RNA
isolation reagents.

RNA purity can be assessed via a spectrophotometer with acceptable A260/ A280 ratios between 1.8 and 2.1.

RNA integrity can be evaluated by analysis on a bioanalyzer. RIN
values should be between 7 and 10. Alternatively, the presence of
discrete bands at 28S and 18S rRNA at an abundance ratio of 2:1
ratio (28S:18S) in denaturing agarose gel electrophoresis indicates
intact RNA.

Both the input RNA and amplified aRNA should be used immediately after purification or stored at ≤-70°C until use. Results may
vary for samples subject to multiple freeze-thaw events.
1. Thaw vials dN, P, FB, D and W. Gently mix, centrifuge (~5 sec)
and place on ice until needed. Briefly centrifuge all tubes containing enzymes immediately prior to use.
2. Place 250 ng to 5 µg of total RNA into the wells of a PCR plate.
Then add 1 µL Promoter Primer (P) to each well.
3. Add an appropriate volume of Nuclease-free Water (W) to the
RNA/Primer mix so that the total volume in each well is 13 µL.
Cover the 96-well plate with a sealing film.
4. Incubate the 96-well plate for 10 minutes at 70°C to denature the
RNA.
NOTE: If there is condensation on the seal cover after the incubation,
centrifuge briefly to collect samples at the bottom of the wells.
Keep the multi-well plate at 4°C until addition of the First Strand
Master Mix in step A6.
5. While denaturing, prepare the First Strand Master Mix by
combining the reagents (Table 4) in a sterile, nuclease-free
microcentrifuge tube. Assemble enough master mix for all samples plus 10% overage to accommodate pipeting error. Gently
mix the reagents by pipetting up and down several times, centrifuge and place the First Strand Master Mix on ice.
Table 4. First Strand Master Mix
Component
First Strand Buffer
DTT
Vial ID
Vol. per Reaction
FB
2.0 µL
D
2.0 µL
dNTP Mix
dN
1.0 µL
Reverse Transcriptase
RT
1.0 µL
I
1.0 µL
RNase Inhibitor
Total
6. While the 96-well plate is incubating at 4°C, add 7 µL of First
Strand Master Mix to each well and mix by pipeting up and down
several times. Cover the 96-well plate with a sealing film.
7. Incubate the 96-plate at 42°C for 2 hours .
NOTE: If there is condensation on the seal cover after the incubation,
centrifuge briefly to collect samples at the bottom of the wells.
8. Stop the reaction by placing the 96-well plate on ice.
7
7.0 µL
4
D. RNA TRANSCRIPT LABELING
B. SECOND STRAND CDNA SYNTHESIS
1. Thaw vials dN, SB and W, mix, briefly centrifuge and keep on ice.
2. Shortly before the completion of the first strand synthesis reaction,
prepare the Second Strand Master Mix (Table 5, below) in a
sterile, nuclease-free microcentrifuge tube. Assemble enough
master mix for all samples plus 10% overage to accommodate
pipeting error. Gently mix the reagents by pipetting up and down
several times, centrifuge and place the Second Strand Master Mix
on ice.
1. Thaw vials RB, B and D. Mix well gently, centrifuge and keep on
ice. Briefly centrifuge the vials containing enzymes immediately
prior to use and place on ice.
2. Prepare the in vitro transcription master mix by combining the
reagents (Table 6, below) in a sterile, nuclease-free microcentrifuge tube. Assemble enough master mix for all samples plus
10% overage to accommodate pipeting error. Gently mix the
reagents by pipetting up and down several times, centrifuge and
place the IVT Master Mix on ice.
Table 5. Second Strand Master Mix
Component
Table 6. In Vitro Transcription (IVT) Master Mix
Vial ID
Vol. per Reaction
Nuclease-free Water
W
106 µL
dNTP Mix
dN
3 µL
Second Strand Buffer
SB
15 µL
DNA Polymerase
DP
5 µL
RNaseH
RH
1 µL
Total
4. Incubate the 96-well plate at 16°C for 2 hours. If using a thermal
cycler, pre-cool to 16°C before starting the reaction.
5. Stop the reaction by placing the 96-well plate on ice.
6. Immediately proceed to purification of the double-stranded cDNA
template.
CDNA
PURIFICATION
In order to proceed to the RNA Transcript Labeling step, purification
of the double-stranded cDNA template is necessary. Use of unpurified cDNA has not been validated with the biotin labeling system
provided in this kit. A suggested protocol for cDNA purification is
outlined in Appendix C.
5
IVT Reaction Buffer
Biotin-Labeled Ribonucleotide
DTT
Vial ID
Vol. per Reaction
RB
6 µL
B
6 µL
D
6 µL
Enhancer Cocktail
EC
6 µL
T7 RNA Polymerase
T7
130 µL
3. Add 130 µL of the Second Strand Master Mix to each well with
sample in it, and mix gently by pipetting up and down several
times. Cover the plate with a sealing film.
C.
Component
3 µL
Total
27 µL
3. At room temperature, add 27 μl of IVT Master Mix to each well
containing sample.
4. Mix gently by pipeting up and down several times. Cover the
plate with a sealing film, then centrifuge briefly to collect the mixture to the bottom of the wells.
5. Immediately place the 96-well plate in a 37°C thermal cycler (or
heating block with heated lid) and incubate for 16 hours. For total
RNA inputs ≥ 2 µg, a 6 hour incubation is sufficient.
6.
Stop the reaction by placing the 96-well plate on ice. If using a
thermal cycler, bring the reaction temperature to 4°C and hold.
7.
Proceed to aRNA purification. Unpurified samples can be stored
at ≤ -70°C for up to 3 days.
E. aRNA PURIFICATION
A suggested protocol for the aRNA purification is provided in
Appendix C.
6
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