| Description |
| The RT² miRNA First Strand Kit provides a
rapid and convenient procedure for efficient universal reverse transcription
from micro RNA. This all-in-one kit has been designed and optimized for
real-time PCR-based miRNA expression analysis with SABiosciences' miRNA qPCR
Arrays and miRNA qPCR Assays. The single tube reaction 1) adds poly-(A)
chain to the 3'-end of the miRNA, 2) primes reverse transcription in an
unbiased manner with an optimized and proprietary sequence tagged with
oligo-d(T), and 3) synthesizes cDNA product with optimal yield. The
resulting proprietary sequence in the cDNA template can only be detected by
SABiosciences' miRNA qPCR Arrays and Assays. The kit also contains a built-in
miRNA External RNA Control (miRNA ERC) to help monitor reverse transcription
efficiency and test for enzyme inhibitors contaminating your RNA samples.
The miRNA ERC is converted into cDNA template and detected by the
appropriate control wells in the miRNA qPCR Arrays. The magnesium,
nucleotide, and other buffer component concentrations are also the most
compatible with RTē SYBR Green qPCR Master Mixes when used for miRNA
expression analysis with miRNA qPCR Arrays and miRNA qPCR Assays. |
| Materials Included |
Please check the kit components
immediately after you receive this package. SABiosciences is not responsible
for any missing items not reported within two (2) business days upon
receipt.
Storage Conditions: This kit is shipped frozen. For long-term storage, keep
entire kit at -20 ºC.
Shelf Life: All reagents are stable for three (3) months after receipt of the kit if
stored at the recommended temperature.
M1: miRNA RT Primer & ERC Mix
M2: 5X miRNA RT Buffer
M3: miRNA RT Enzyme Mix
100 mM DTT
RNase-free H2O |
| Brief Protocol: For Experienced
Users |
First-time Users should refer to the RT²
miRNA PCR Array System or the RT²
miRNA qPCR Assay User Manuals.
- For each RNA sample, combine the following in a sterile PCR tube:
| Enriched Small RNA
50 ng to 200 ng |
| miRNA RT Primer & ERC Mix (M1) |
1.0 |
µL |
| 5X miRNA RT Buffer (M2) |
2.0 |
µL |
| miRNA RT Enzyme Mix (M3) |
1.0 |
µL |
| 100 mM DTT |
1.0 |
µL |
|
RNase-free H2O to a final
volume of
|
10.0 |
µL |
-
Mix the contents gently with a pipettor followed by brief centrifugation.
-
Incubate at 37 ºC for 2 hours.
-
Heat at 95 ºC for 5 minutes to degrade the RNA and to inactivate the reverse
transcriptase.
-
Chill on ice for at least one minute and add 90 µL of RNase-free H2O to each
10-ml of cDNA synthesis reaction. Mix well.
-
Hold the finished First Strand cDNA Synthesis Reaction on ice until the next
step or store overnight at -20 ºC.
|
