Design of a new plasma external reference and analysis of nucleic acid detection

Abstract

Objective To design a novel plasma external reference (PLACON) to detect and analyze human plasma mRNA and miRNA. Methods A conserved sequence was selected from the genome of the low-level species Caenorhabditis elegans. It was designed as a novel plasma external reference and BLAST was used to detect the specificity of PLACON sequences. Plasma samples of patients with malignant tumors and cancer cell supernatants were collected. And PLACON, β-actin, GAPDH, and U6 which were added to the plasma or cell supernatant respectively, were used for amplification comparison to detect plasma mRNA, miRNA and cell supernatant mRNA, by quantitative real-time PCR (RT-qPCR). Results BLAST detected that the PLACON sequence had good specificity and no cross-correlation with the human genome. Melting curves showed high specificity in RT-qPCR reaction. The cycle threshold values of mRNA levels were significantly lower in plasma of PLACON group than the GAPDH and β-actin groups (P < 0.01). The cycle threshold (CT) value of miRNA level amplification in plasma in PLACON group was significantly lower than that in U6 group (P < 0.01). PLACON was validated in mRNA amplification experiments in cancer cell supernatants, and similar results were obtained. Conclusion PLACON sequence is specific, and shows better amplification advantage as internal reference in plasma, with high stability and efficient amplification. It can be used for quantitative detection of plasma mRNA and miRNA.

Keywords

• external reference
• internal reference
• rt-qpcr
• mirna in plasma