Total RNA was purified using the miRNeasy Mini Kit from [A] 2 identical HeLa S3 cell pellets with identical passage numbers and [B] 2 HeLa S3 cell pellets collected from different parental stocks at different times. Using the miScript PCR System, cDNA was prepared and used as a template in real-time PCR with the miFinder miScript miRNA PCR Array. Plotting CT values of the replicates against each other demonstrated the high technical and biological reproducibility of the results. These experiments were performed by a first-time user of the miScript PCR System.
In a reverse transcription reaction with miScript HiSpec Buffer, mature miRNAs are polyadenylated by poly(A) polymerase and converted into cDNA by reverse transcriptase with oligo-dT priming. The cDNA is then used for real-time PCR quantification of mature miRNA expression.
In a reverse transcription reaction with miScript HiFlex Buffer, miRNAs and other noncoding RNAs (ncRNAs) are polyadenylated by poly(A) polymerase and converted into cDNA by reverse transcriptase with oligo-dT priming. mRNAs are converted into cDNA by reverse transcriptase using both oligo-dT and random priming. Detection of mature miRNA, precursor miRNA, other ncRNA, and mRNA can be performed using the appropriate assays.
Two buffers are supplied with the miScript II RT Kit. Use miScript HiSpec Buffer for cDNA synthesis to enable either mature miRNA profiling (using miScript miRNA PCR Arrays) or mature miRNA quantification using individual miScript Primer Assays. Use miScript HiFlex Buffer for cDNA synthesis to enable quantification of mature miRNA, precursor miRNA, noncoding RNA (ncRNA), and/or mRNA from the same cDNA. cDNA prepared using either miScript HiSpec Buffer or miScript HiFlex Buffer can be used with miScript PCR Controls.