Total RNA was purified from HeLa S3 cells using the miRNeasy Mini Kit. cDNA was prepared from a range of RNA amounts from 10 pg to 2 µg using the miScript II RT Kit with [A] miScript HiSpec Buffer or [B] miScript HiFlex Buffer (both provided in the kit). Each cDNA was used as a template in real-time PCR reactions using a miScript Primer Assays for miR-16, miR-20a, or miR-21. Linear standard curves demonstrated the broad dynamic detection range and high amplification efficiency of the miScript PCR System.
Synthetic miR-21 was used to generate cDNA using the miScript II RT Kit with [A] miScript HiSpec Buffer or [B] miScript HiFlex Buffer (both provided in the kit). A range of amounts from 10 copies to 106 copies of this cDNA was used in real-time PCR using the miScript PCR System. Real-time PCR was performed on the Rotor-Gene Q cycler. The resultant CT values decreased linearly with increasing miRNA copy number, indicating sensitive detection from a wide range of template amounts.