Enriched high­throughput reverse transcription­quantitative PCR template preparation without pre­amplification.

A cDNA template with a high concentration is required to generate a high number of copies for accurate downstream high­throughput reverse transcription­quantitative PCR screening. However, with the traditional method, pre­amplification is not widely available. In the present study, a novel strategy to resolve the pre­amplification limitation has been developed. Total RNA was extracted using a commercially available RNeasy Micro kit then, the cDNA was synthesized using SuperScript® III First­Strand Synthesis system. PCR inhibitors (proteins and soluble salt ions) in the enriched cDNA were removed using saturated phenol­chloroform 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 phenol­chloroform extraction, and the positive detection rate increased to 97.04%. Notably, the positive detection rate of cDNA prepared using this method of high­throughput 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.