| Description |
| The RT² First Strand Kit provides a rapid and convenient procedure for efficient first strand cDNA synthesis. The kit also contains an effective genomic DNA elimination step and a built-in External RNA Control. This all-in-one kit has been designed and optimized for real-time PCR-based gene expression analysis with SABiosciences' RT² Profiler™ PCR Arrays and RT² qPCR Primer Assays. The kit includes a proprietary procedure to effectively eliminate contaminating genomic DNA from RNA samples before reverse transcription. Random hexamers and oligo-dT prime reverse transcription in an unbiased manner, and a reverse transcriptase synthesizes cDNA product with optimal yield and length. A built-in External RNA Control helps monitor reverse transcription efficiency and test for enzyme inhibitors contaminating your RNA samples when used together with RT² RNA QC PCR Array and RT² Profiler™ PCR Array. The magnesium and nucleotide concentrations and other buffer components are the most compatible with RT² SYBR Green qPCR Master Mixes when used in gene expression analysis with RT² Profiler™ PCR Arrays and RT² qPCR Primer Assays. |
| Materials Included /
Packing List |
|
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 following kit is shipped frozen. For long-term storage, keep entire
box at -20º C
Shelf Life: All reagents are
stable for 6 months after receipt of the kit if stored at the recommended
temperature.
GE: 5X gDNA Elimination Buffer
BC3: 5X Reverse Transcription Buffer 3
H2O: RNase-free H2O
P2: Primer and External Control Mix
RE3: RT Enzyme Mix 3
|
| Brief Protocol |
- Prepare a separate Genomic DNA Elimination mixture for each RNA sample:
|
Total RNA |
25.0 ng to 5.0 |
µg |
|
|
GE (5X gDNA Elimination Buffer) |
2.0 |
µl |
|
|
RNase-free H2O to a final volume of |
10.0
|
µl |
|
We recommend starting with 0.5 to 1.0 µg of total RNA to maximize the positive call rate.
Use a consistent amount of RNA for each sample.
-
Incubate at 42 ºC for 5 min, and immediately place on ice for at least 1
minute.
- Generate the following RT cocktail scaling up the recipe for the number of samples and viscosity pipetting errors:
| BC3 (5X RT
Buffer 3) |
4
µl |
| P2 (Primer and External Control
Mix) |
1
µl |
| RE3 (RT Enzyme
Mix 3) |
2 µl |
| H2O RNase-free |
3 µl |
| Final Volume |
10
µl |
-
Briefly centrifuge all mixtures and the RT cocktail to the bottom of the tube.
- Add 10 µl of the RT Cocktail to each 10-µl Genomic DNA
Elimination Mixture for a final volume of 20 µl.
- Incubate at 42 ºC for exactly 15 min
and then immediately stop the reaction by heating at 95 ºC
for 5 minutes.
- Hold the finished reaction on ice until ready to use for real-time PCR, or
place at -20 ºC for long-term storage.
|
