miRNA First Strand cDNA Synthesis Kit (Add Tail)
Cat. No.: SB-G3334-25
Size: 25 rxns
Description
MicroRNAs(miRNAs) are members of a rapidly growing class of small non-coding RNAs, 19-23 nt in length, known to contribute to the regulation of gene expression in plants and animals. miRNA has been subsequently shown to be encoded in the genomes of higher vertebrates, including humans ans animals.
This miRNA First Strand CDNA Synthesis Kit is used to converting miRNAs, and other molecules, into cDNA to enable specific RNAs to be quantified by real-time PCR. In a single-tube reaction,RNA molecules are polyadenylated and reverse transcribed using poly(A) polymerase and our optimized MMLV reverse transcriptase, both of which are included in the miRNA RT Enzyme Mix. The Kit also contains U6 reference primers, including U6 Forward PCR Primer and Universal Reverse PCR Primer suitable for human, rat, and mouse, for subsequently PCR to check cDNA synthesis. For target gene PCR or qPCR, the specific forward primer (design as desired) and Universal Reverse PCR Primer are needed.
Storage and Handling Conditions
Wet ice bag transportation; Stored at -20℃ temperature, valid for 12 months.
Component
| Component Number |
Component |
G3334-25 |
| G3334-1 |
miRNA RT Enzyme Mixa |
25 μL |
| G3334-2 |
5×Reaction Bufferb |
100 μL |
| G3334-3 |
U6 Forward PCR Primer (10 μM) |
100 μL |
| G3334-4 |
Universal Reverse PCR Primer (10 μM) |
100 μL |
| G3334-5 |
Nuclease-Free Water |
1 mL |
| Product Manual |
1 |
a：with RNase Inhibitor and Poly(A) Polymerase
b：with reverse transcription primers, dNTP Mixture, ATP and Mg²+
Assay Protocol
1. Preparation of reverse transcription reaction system (20 μL);
| Component |
Volume |
| 5×Reaction Buffer |
4 μL |
| miRNA RT Enzyme Mix |
1 μL |
| Total RNA/miRNAa |
x μL |
| Nuclease-Free Water |
Add to 20 μL |
a: the recommended total RNA is 2 μg (no more than 5 μg), and the recommended miRNA is 200 ng.
2. Gently mix and centrifuge;
3. poly(A) polymerize and Reverse transcription program setting:
| Temperature |
Time |
| 37℃ |
60 min |
| 85℃ |
5 s |
4. The obtained cDNA product is recommended to dilute before subsequently PCR or qPCR.
Note:
1. Please wear disposable gloves during operation to avoid RNase contamination.
2. The reverse transcription products can be stored at -20℃ for a period. If long-term storage is needed, it is recommended to store them at -80℃ after packing and avoid repeated freeze-thaw cycles.
