Overview
The Reliance Select cDNA Synthesis Kit demonstrates an efficiency superior to that of the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific Inc. #18091200) in the presence of inhibitors. Varying amounts of Cervical Adenocarcinoma (HeLa-S3) Total RNA (Thermo Fisher Scientific Inc. #AM7852), ranging from 1 pg to 2 μg, were used in a Reliance Select Kit (
) or a SuperScript IV System () reaction with oligo(dT)20 according to the corresponding protocols. One of each duplicate reaction was spiked with a known PCR inhibitor. First-strand cDNA libraries were used in qPCR reactions using the SsoAdvanced SYBR® Green Supermix and an assay panel that included high- and low-expressing targets, GC-rich amplicons, and amplicons of varying lengths. The Reliance Select Kit delivers more consistent results and earlier average Cq (quantification cycle) than the SuperScript IV System, as is evident from the lower ΔCq values of treated versus untreated reactions. Cqs in the SuperScript IV System reactions were delayed (up to a 3-Cq delay with formalin) in the presence of most inhibitors, whereas Reliance Select Kit reactions were within 0.5 Cq of their controls.
The Reliance Select Kit delivers earlier Cqs (quantification cycles) compared to the SuperScript IV System when tested with artificially degraded RNA samples. Varying amounts of degraded Cervical Adenocarcinoma (HeLa-S3) Total RNA ranging from 1 ng to 1 μg, were reverse transcribed according to the product protocols, using the Reliance Select Kit () or the SuperScript IV System () reactions and random primers. First-strand cDNA libraries were used in qPCR reactions with the SsoAdvanced SYBR® Green Supermix and an assay panel that included common reference gene targets. The average Cq across the concentration range for a given target using degraded RNA was compared to the average Cq of duplicate reactions using intact RNA. The Reliance Select Kit delivered more accurate results between intact and degraded RNAs than the SuperScript IV System, as is evident from the lower ΔCq values.
Reliance Select Kit allows for sensitive detection of low-abundance targets. ERCC RNA Spike-In Mix controls (Thermo Fisher Scientific #4456740) were reverse transcribed in the presence or absence of background RNA (1 μg of Cervical Adenocarcinoma [HeLa-S3] Total RNA) using random primers and the Reliance Select Kit or SuperScript IV System according to the product protocols. First-strand cDNA libraries were used in qPCR reactions targeting ERCC-0034. The low-abundance ERCC spike-ins in the presence of background RNA were more sensitively detected with the Reliance Select Kit. This is evident when comparing the <1 Cq shift between samples with and without 1 μg background RNA when transcribed with the Reliance Select Kit to the >3 Cq delay when transcribed with the SuperScript IV System. RFU, relative fluorescence units.
