Description
The EZBioscience ® microRNA Reverse Transcription Kit PLUS uses the method of adding poly A tail to the 3' end of miRNA by the E. coli Poly(A) Polymerase to synthesize the first strand cDNA of the miRNA. This kit contains U6 forward primer (U6 primer), and Universal 3'qPCR Primer that can be used for the quantitative detection of all miRNAs including U6. So only a specific forward primer for each target miRNA is needed for qPCR reactions. This Kit includes two steps: first, The RNAs are treated with gDNA Remover to eliminate the genomic DNA. Then, poly(A) tail is added to the 3’-end of miRNA molecules in the poly(A) tailing reaction. The reverse transcription reaction is performed using a specially designed Universal RT Primer to generate the first strand of the miRNA.
The Kit is a rapid reverse transcription kit containing high-efficiency DNase for the removal of genomic DNA. It contains fIve tubes of reagents: gDNA Remover, miRNA RT Enzyme Mix, 4× miRNA RT Buffer, Universal 3’qPCR Primer and U6 Primer.
Among them, gDNA Remover mainly includes concentrated DNase and buffer. It only needs to react at room temperature (19 ~ 27°C) for 5 minutes to degrade more than 95% of residual genomic DNA, which greatly reduces the interference to the results.
The miRNA RT Enzyme Mix mainly contains E. coli Poly(A) Polymerase, reverse transcriptase, and RNase Inhibitor. The E. coli Poly(A) Polymerase in it not only has high efficiency of adding poly(A) tail, but also specifically recognizes single-stranded miRNA, thus avoiding further reverse transcription reaction of miRNA precursor with double-stranded structure; The mutant M-MLV reverse transcriptase has strong anti-interference ability and amplification ability, and the amplification efficiency is particularly good.
The 4× miRNA RT Buffer contains all the raw materials and primers for miRNA polyadenylation and reverse transcription reaction, including Oligo (dT)-universal tag primers, buffer and dNTPs, and has been carefully optimized to ensure polyadenylation and the reverse transcription are simultaneously performed efficiently.
Components
Components | EZB-miRT2-plus-S (20 Rxns) | EZB-miRT2-plus-L (50 Rxns) |
gDNA Remover | 22 μl | 55 μl |
miRNA RT Enzyme Mix | 44 μl | 110 μl |
4× miRNA RT Buffer | 110 μl | 275 μl |
Universal 3’ qPCR Primer | 300 μl | 750 μl |
U6 Primer | 60 μl | 150 μl |
Nuclease free ddH2O | 1 ml | 1 ml |
Storage
Store at -20°C.
Features
1. Fast and simple procedure: This kit uses the method of Poly (A) tailing reaction and reverse transcription reaction in a single step assay to synthesize the first strand cDNA of miRNAs. There is no need to design additional reverse transcription primers. high-quality cDNA containing all miRNAs can be obtained in 30 minutes.
2. High specificity: Poly(A) Polymerase not only has high A-tailing efficiency, but also specifically recognizes mature single-stranded miRNA, thus avoiding the reverse transcription reaction of miRNA precursors with double-stranded structure.
3. High efficiency: This kit uses an optimized combination of high-efficiency Poly(A) Polymerase and reverse transcriptase, which can efficiently carry out Poly (A) tailing and reverse transcription reactions. Compared with common commercial reverse transcription products, this kit reverses cDNA with the smallest quantitative CT value and has excellent reverse transcription efficiency.
4. The kit contains the Universal 3’qPCR Primer that is the qPCR reverse primer for all miRNAs including U6, and the U6 Primer that is the qPCR forward primer for U6, which are then used in real-time qPCR, along with your miRNA-specific 5’ primer(s), to quantify specific miRNA sequences in the cDNA.
For detailed information, please check the product manual.
