The EZBioscience® 4× microRNA Reverse Transcription Mix is based on the Stem-loop method to synthesis the first strand of cDNA for miRNA. The kit contains a genomic DNA removal step, which can remove the contamination of genomic DNA by reacting at room temperature for 5 min, which ensures the subsequent experimental results are more accurate.
The kit contains two tubes of reagents: gDNA Remover and 4× miRNA RT Mix. gDNA Remover mainly includes concentrated DNase and buffer; 4× miRNA RT Mix mainly contains reverse transcriptase, buffer, RNase Inhibitor, dNTPs required for reverse transcription except reverse primer. The first strand of cDNA for miRNA can be rapidly synthesized by designing a specific reverse transcription primer for the target miRNA based on the sequence of the target miRNA and the Stem- loop sequence provided by this kit.
The Reverse transcriptase in the mix is a genetic engineered enzyme based on M-MLV (RNase H-) reverse transcriptase. The reverse transcriptase lacking RNase H activity is suitable for preparing cDNA. And the multiple site-mutations of Reverse transcriptase can obviously increase its affinity to miRNA template and its strand extending ability, which make reverse transcription reaction more efficient. Moreover, this transcriptase is rather resistant to common reverse transcriptase inhibitors. This product is also very suitable for reverse transcription using plant tissue miRNA.
4× miRNA RT Mix
Nuclease free ddH2O
1 ml × 2 tubes
Store at -20°C.
Stem-loop primer design
A specific Stem-loop RT primer is recommended for each miRNA by using this Kit. One suggested universal stem-loop primer sequence is as follows: 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACT-GGATACGAC-3’. Thus, for each miRNA, the RT primer sequence should be like this: 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACT-GGATACGAC + miRNA RCS (6 ~ 8 nt of reverse complementary sequence for each miRNA, from 3’ end of the mature miRNA sequence)-3’. [The mature miRNA sequence could be got from the NCBI gene database, or miRbase].
1. In each sample, reverse transcription of each target miRNA and internal reference gene to be tested respectively.
2. The reverse transcription primers need to be designed for each target miRNA and internal reference gene to be tested.
For detailed information, please check the product manual.