Introduction
The EZBioscience® EZ-press Cell to cDNA Kit for microRNA is directly used for reverse transcription fast and conveniently from cultured cell lysates. The cells are first lysed in the Cell Lysis Buffer to release the microRNA. Then, cell lysates are treated with gDNA Remover to eliminate the contaminating genomic DNA. Finally, Polyadenylation and Reverse transcription are completed in one step in the same reaction system.
The kit contains 6 tubes of reagents: Cell Lysis Buffer, gDNA Remover, miRNA RT Enzyme Mix, 4× miRNA RT Buffer, a forward U6 primer, and a universal qPCR reverse primer (Universal 3' qPCR Primer), which can be used for the quantitative detection of all miRNAs. So only a specific forward primer for each target miRNA is needed for qPCR reactions.
The miRNA RT Enzyme Mix mainly contains E.coli Poly(A) Polymerase, reverse transcriptase, and RNase Inhibitor. Among them, E.coli Poly(A) Polymerase not only has high efficiency of adding poly(A) tail, but also specifically recognizes single-stranded mature miRNA, thus avoiding further reverse transcription reaction of miRNA precursor with double-stranded structure. The M-MLV mutant reverse transcriptase has strong anti-interference ability and amplification ability, and the amplification efficiency is particularly good.
4× miRNA RT Buffer contains all the raw materials and primers for miRNA polyadenylation and reverse transcription reaction, including Oligo (dT)-universal tag primer, buffer and dNTPs, and has been carefully optimized to ensure polyadenylation and the reverse transcription are simultaneously performed efficiently.
Components
Components | EZB-miRT1 (50 Rxns) |
Cell Lysis Buffer | 11 ml |
gDNA Remover | 110 μl |
miRNA RT Enzyme Mix | 110 μl |
4× miRNA RT Buffer | 275 μl |
Universal 3’ qPCR Primer (10 μM) | 750 μl |
U6 Primer (10 μM ) | 150 μl |
Nuclease free ddH2O | 1 ml |
Storage
Store at -20°C.
Experimental Procedure at a Glance
For detailed information, please check the product manual.
