miScript Reverse Transcription Kit (50)

QIAGEN's miScript Reverse Transcription Kit converts RNA preparations containing microRNA (miRNA) into cDNA for use in real-time PCR quantification within the miScript PCR System, which also includes the miScript SYBR Green PCR Kit and miScript Primer Assays for miRNA detection and quantification. According to the system's handbook, each reverse-transcription reaction is assembled with 4 µl of 5x miScript RT Buffer, 1 µl of miScript Reverse Transcriptase Mix, variable RNase-free water, and up to 1 µg of template RNA in a total volume of 20 µl. The reaction is incubated for 60 min at 37 °C, followed by 5 min at 95 °C to inactivate the enzyme.

The kit is recommended for use with total RNA preparations that include small RNAs. QIAGEN suggests purification with miRNeasy Kits together with an on-column DNase digestion step to remove traces of genomic DNA, which is especially relevant for precursor miRNA quantification because of the low abundance of these targets. The resulting cDNA supports parallel quantification of mature and precursor miRNA in the same experiment, with typical inputs of 1–3 ng cDNA per reaction for mature miRNA and 10–20 ng per reaction for precursor miRNA, run with QuantiTect SYBR Green PCR Master Mix at an optimized 2.5 mM Mg²⁺ concentration. For accurate relative quantification, miScript PCR Controls provide primer assays against small nucleolar and small nuclear RNAs designed with sequence homology across human, mouse, and rat, allowing the same control to be used across all three species. Cycling conditions optimized for miRNA and noncoding RNA quantification typically yield PCR products with melting temperatures of approximately 74–77 °C. Workflow applications enabled by the system include miRNA expression analysis, RT-qPCR validation, and profiling studies.