Enriched high‑throughput reverse transcription‑quantitative PCR template preparation without pre‑amplification

Abstract

a cdna template with a high concentration is required to generate a high number of copies for accurate downstream high-throughput reverse transcription-quantitative Pcr screening. However, with the traditional method, pre-amplification is not widely available. In the present study, a novel strategy to resolve the pre-amplification limitation has been developed. Total RNA was extracted using a commercially available RNeasy Micro kit then, the cDNA was synthesized using SuperScript ® III First-Strand Synthesis system. PCR inhibitors (proteins and soluble salt ions) in the enriched cdna were removed using saturated phenol-chloroform extraction. Finally, genes were evaluated using PCR amplification and the BioMark™ HD system. The positive detection rate of individual target gene expression reached 70.18%; however, it markedly decreased to 35.42% using PCR amplification without prior dilution. next, the reverse transcription product was purified using saturated phenol-chloroform extraction, and the positive detection rate increased to 97.04%. Notably, the positive detection rate of cdna prepared using this method of high-throughput and traditional Pcr (97.04 vs. 96.6%) was not significantly different. In conclusion, the results demonstrate the novel method was an easy and reproducible method for performing robust and highly accurate targeted amplification.

Heterogeneity induced GZMA-F2R communication inefficient impairs antitumor immunotherapy of PD-1 mAb through JAK2/STAT1 signal suppression in hepatocellular carcinoma

“…Peripheral blood mononuclear cells (PBMCs) were separated as described in a previous report [ 50 ]. Briefly, peripheral blood was collected in a 15-ml tube and centrifuged at 2000 rpm.…”