Overview

Norgen's microScript microRNA cDNA Synthesis Kit is an all-in-one, ready-to-use product for the reverse transcription of microRNA from either Total RNA preparations or enriched microRNA preparations. The kit contains the 2x Reaction Mix and the microScript microRNA Enzyme Mix. The kit utilizes Norgen’s microScript Reverse Transcriptase, a mutant version of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase. It has reduced RNase H activity and increased thermal stability.

The workflow of Norgen's microScript microRNA cDNA Synthesis Kit involves a simple, single-tube set-up by the mixing of 2x Reaction Mix, Enzyme Mix and the RNA template. The reaction can then be carried out in a thermocycler. A poly (A) tail is first added to the RNA template, followed by cDNA synthesis using an adapter primer. In addition to the ease-of-use, the single-tube set-up provides superb consistency and sensitivity. The cDNA could be used in a PCR or qPCR amplification using a Universal PCR Reverse Primer and the forward primer that contains the sequence of the microRNA of interest. A single cDNA preparation could be used for PCR amplification of a number of different microRNAs. In addition, the cDNA preparation could be used for PCR or qPCR detection (using gene-specific forward and reverse primers) of mRNA or large RNA if total RNA preparation was the starting template. This could allow for parallel evaluation of expression level of microRNAs and microRNA-targets.

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Supporting Data

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Figure 1. High Sensitivity with Robust Amplification of microRNAs from Different Amounts of RNA Input using Norgen’s microScript microRNA cDNA Synthesis Kit. Norgen’s microScript microRNA cDNA Synthesis Kit was used in an RT-qPCR reaction to amplify the human miR-1246 and miR-21 from a dilution series of HeLa total RNA isolated using Norgen’s Total RNA Purification Kit. The PCR target was robustly amplified from a broad range of RNA inputs, from the 200 ng down to 2 pg.

Figure 2. Robust Reverse Transcription of All RNA Transcripts (microRNAs and mRNA). Norgen’s microScript microRNA cDNA Synthesis Kit could effectively reverse transcribe both microRNA and other RNA species such as mRNA and large RNAs, allowing the parallel study of microRNA and its targets. HeLa RNA (200 ng) was used as a template in a reverse transcription using the microScript microRNA cDNA Synthesis Kit. The resulting cDNA was used in three different qPCR reactions for three different species of RNA. The top three panels showed the successful PCR amplification curve for miR-21 (microRNA), 5S rRNA (RNA without poly-A tail) and S15 (large mRNA). The lower three panels showed that each of the three PCR amplification showed unique amplicon with unique melt curve characteristics.

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