microRNA RT Master Mix for qPCR Ⅲ (poly A)
Based on 3 publication(s) in Google Scholar
MCE microRNA RT Master Mix for qPCR Ⅲ (poly A) uses poly(A)-tailing method to perform reverse transcription from miRNA first-strand to cDNA.
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Storage :
-20°C, 2 years
Avoid repetitive freeze-thaw cycles
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Description & Advantages
MicroRNAs are a class of non-coding RNAs with a length of about 22 nt, which play an important role in the regulation of gene expression in plants and animals.
MCE microRNA RT Master Mix for qPCR Ⅲ (poly A) uses poly(A)-tailing method to perform reverse transcription from miRNA first-strand to cDNA. The kit contains poly(A) polymerase (PAP) for the miRNA 3' end poly(A) reaction and all the raw materials and primers for miRNA reverse transcription reaction. It ensures that poly(A) modification process and reverse transcription process can be performed simultaneously and efficiently in miRNA 3' end. Only design miRNA-specific forward primers and use them together with the universal reverse primers included in the kit for subsequent qPCR experiments. Additionally, this kit provides universal U6 internal forward/reverse primers applicable for human, rat, and mouse that can be used for plotting standard curves.
Protocol
1. miRNA Poly(A) tailing and reverse transcription
Mix all reagents thoroughly and briefly centrifuge. Prepare the reaction on ice as follows:
| Component | 10 μL reaction |
|---|---|
| 2× miRNA RT Buffer | 5 μL |
| miRNA RT Mix | 1.75 μL |
| Total RNA | X μL |
| RNase-Free H2O | To 10 μL |
Note: Avoid repeated freeze-thawing of RNA and RNase contamination. Adjust template volume according to RNA concentration and keep the starting RNA amount consistent among samples.
Run the following program:
| Step | Temperature | Time |
|---|---|---|
| Poly(A) tailing and reverse transcription | 37°C | 60 min |
| Enzyme inactivation | 85°C | 5 min |
| Hold | 4°C | Hold |
The resulting cDNA may be used directly for qPCR or stored at -20°C.
2. miRNA qPCR
For each miRNA, design only the miRNA-specific Forward Primer and use it with the Universal Reverse Primer supplied in the kit. A recommended 20 μL reaction is shown below:
| Component | 20 μL qPCR reaction |
|---|---|
| SYBR Green qPCR Master Mix (2×) | 10 μL |
| miRNA-specific Forward Primer (10 μM) | 0.4 μL |
| Universal Reverse Primer (10 μM) | 0.4 μL |
| RT product | 2 μL |
| RNase-Free H2O | 7.2 μL |
qPCR may be performed with initial denaturation at 95°C followed by 40 cycles of denaturation at 95°C and annealing/extension at 60°C, followed by melting-curve analysis. U6 may be detected using the supplied U6 Forward Primer and U6 Reverse Primer as an internal reference.
Publications
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Journal Impact Factor
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Most Recent
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Am J TransplantBrg1 interacts with liver X receptor and H3K18la to regulate ferroptosis via the CYP4A14/20-HETE axis in liver transplantation. [Abstract]2026 Mar 27:S1600-6135(26)00166-8. PMID: 41903856
Storage
-20°C, 2 years
Avoid repetitive freeze-thaw cycles
Components
| Components | HY-K0512-10 rxns | HY-K0512-50 rxns |
|---|---|---|
| miRNA RT Mix | 17.5 μL | 87.5 μL |
| 2 × miRNA RT Buffer | 50 μL | 250 μL |
| Universal Reverse Primer (10 μM) | 800 μL | 4 mL |
| U6 Forward Primer (10 μM) | 100 μL | 500 μL |
| U6 Reverse Primer (10 μM) | 100 μL | 500 μL |
| RNase-Free H2O | 2 mL | 10 mL |
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