Why does the miScript PCR System include a Universal Primer?
The principle of the miScript PCR System is shown in the figure “miScript principle”. Unlike mRNAs, miRNAs are not polyadenylated in nature. During the reverse-transcription step, miRNAs are polyadenylated by poly(A) polymerase. Reverse transcriptase converts RNA to cDNA using oligo-dT and random primers. Polyadenylation and reverse transcription are performed in parallel in the same tube. The oligo-dT primers have a universal tag sequence on the 5′ end. This universal tag allows amplification in the real-time PCR step. The cDNA serves as the template for real-time PCR analysis using the miScript Primer Assay in combination with the miScript SYBR Green PCR Kit. miRNAs are amplified using the miScript Universal Primer, which primes from the universal tag sequence, together with the miRNA-specific primer (the miScript Primer Assay).
