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

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.