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Home > Products > PCR Home > PCR Array > RT² FFPE PreAMP cDNA Synthesis Kit
RT² FFPE PreAMP cDNA Synthesis Kit
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| Description |
| The RT² FFPE PreAMP cDNA Synthesis Kit is
intended for the synthesis and pre-amplification of cDNA from FFPE RNA
samples prior to performing RT² Profiler™ PCR Array-based analysis.
The kit contains the components for the first strand cDNA synthesis, and RT²
PreAMP PCR Master Mix which, together with the pathway-specific RT² PreAMP
cDNA Primer Mix, employs multiplex PCR-based pre-amplification to provide
amplification of 89 gene-specific cDNA target templates with minimal bias
for analysis with a pathway-focused RT² Profiler™ PCR Array. The first
strand cDNA synthesis components of this kit contain all the reagents
required for the reverse transcription of RNA. The RT² PreAMP PCR Master Mix
(2X) contains all the reagents and buffers required for the multiplex PCR-based
pre-amplification: PCR buffer formulated for multiplex PCR, a
high-performance HotStart Taq DNA polymerase and nucleotides. The Side
Reaction Reducer that comes with the kit minimizes any potential nonspecific
reactions in the downstream PCR Array applications. After performing the
reverse transcription step, simply add the master mix to PCR tubes along
with your cDNA template and the pathway-specific RT² PreAMP cDNA Primer Mix
for a specific PCR Array and perform multiplex PCR for 8 cycles on any
end-point thermal cycler. Then treat the amplified samples with the Side
Reaction Reducer, and the samples will be ready for use in the RT² Profiler
PCR Arrays. The cDNA product from each first strand synthesis reaction is
sufficient for performing the pre-amplification step for up to four
different pathways. |
| Materials Included /
Packing List |
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Please check the kit components immediately after you receive this
package. SABiosciences is not responsible for missing items not reported within
two (2) business days upon receipt.
Storage Conditions: The
kit is shipped on dry ice or cold packs. For long-term storage, keep at -20
ºC. If entire volume is not be used all at once, divide into aliquots and
store at -20 ºC. Avoid repeated freezing and thawing.
Shelf Life: All reagents are
stable for 6 months after receipt of the kit if stored at the recommended
temperature.
RT² FFPE PreAMP cDNA Synthesis Kit (Catalogue # C-07) A:
First Strand cDNA Synthesis Components (enough for 12 20-µl RT reactions) -
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One (1) tube of GE (5X gDNA Elimination Buffer)
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One (1) tube of BC3 (5X Reverse Transcription Buffer 3)
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One (1) tube of RE (cDNA Synthesis Enzyme Mix)
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One (1) tube of RI (RNase Inhibitor)
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One (1) tube of P2 (Primer and External Control Mix)
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One (1) tube of RNase-free H2O
B: FFPE PreAMP components
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One (1) tube of PA-030 (RT² PreAMP PCR Master Mix) containing 600µl of 2X
solution and enough for 4 25-µl standard reactions for each of the 12
cDNA synthesized (48 pre-amplification reactions)
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One (1) tube containing 96µl Side Reaction Reducer (SR1) enough for
4 standard reactions for each of 12 samples (48 pre-amplification
reactions)
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| Brief Protocol |
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Read carefully the User Manual of the RT² FFPE PreAMP cDNA Synthesis Kit
prior to the first-time use. Refer to the instructions in the User Manual
every time you use the RT² FFPE PreAMP cDNA Synthesis Kit (catalog #
C-07).
Ensure that you do not contaminate the RT² FFPE PreAMP Primer Mix by using a fresh
pipette tip every time you draw an aliquot from the tube.
- Perform first-strand cDNA synthesis using the RT² FFPE PreAMP cDNA
Synthesis Kit as follows:
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Add 2 µl of GE to 8 µl of RNA (100ng - 1µg).
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Incubate at 37 ºC for 15 min and immediately chill on ice.
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Mix a master mix for the RT reaction as below:
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For 1 reaction: |
| BC3 |
4 µl |
| RE |
1 µl |
| RI |
1 µl |
| P2 |
1 µl |
| RNase-free H2O |
3 µl |
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Add 10 µl of the RT master mix to 10 µl GE-treated
RNA.
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Incubate at 42 ºC for 30 min and heat at 95 ºC for 5 min.
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Chill on ice or store at -20 ºC until use.
- For the normal standard reaction, mix the following components in a PCR
tube:
| 12.5 µl |
2X RT² PreAMP PCR Master Mix (PA-030) |
| 7.5 µl |
1 µl RT² FFPE PreAMP Primer Mix for the RT²
Profiler™ PCR Array of your choice |
| 5 µl |
Template: undiluted cDNA from a 20-µl
first strand synthesis reaction |
| 25.0 µl |
final volume |
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Perform 8 cycles of PCR in a thermal cycler:
NOTE: The 10 min step at 95 ºC is required to activate the HotStart
Taq DNA polymerase.
95 ºC, 10 min; 8 cycles of (95 ºC, 15 sec; and 60 ºC, 2 min); 4 ºC
forever
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Add 2 µl of the Side Reaction Reducer (SR1) to each pre-amplified
reaction, and incubate at 37 ºC for 15 min followed by heat inactivation
at 95 ºC for 5 min.
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Dilute the 27-µl pre-amplified templates to 111 µl by adding 84 µl
of dd H2O. Use immediately and keep on ice prior to loading onto
the RT² Profiler™ PCR Array or store at -20 ºC until use.
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For use in the RT² Profiler™ PCR Array, mix well the following components in
15-mL conical tube:
| 1275 |
µl |
2X SABiosciences RT² qPCR SYBR Green Master Mix (Note:
Use the appropriate master mix specific for your real-time PCR
instrument) |
| 102 |
µl |
diluted PreAMP PCR reaction (from Step 5) |
| 1173 |
µl |
ddH2O |
| 2550 |
µl |
final volume |
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Add 25 µl of the above Experimental Cocktail to each well of the PCR Array,
preferably from a reservoir with an eight-channel pipettor (or a
twelve-channel pipettor but only using eight tips).
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Run the following real-time thermal cycler program:
NOTE: The 10 min step at 95 ºC is required to activate the HotStart Taq DNA
polymerase.
95 ºC, 10 min; 40 cycles of (95 ºC, 15 sec; and 60 ºC, 60 sec)
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Program the real-time thermal cycler to detect and record the SYBRŪ Green I
signal from every reaction during the annealing step of each cycle.
SYBRŪ is a registered trademark of Molecular Probes, Inc.
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