Establishment and validation of hsa_circ_0004123 absolute quantitative PCR method / 解放军医学杂志

ABSTRACT

Objective To establish an absolute reverse transcription fluorescence quantitative PCR method (RT-qPCR) that can accurately quantify the circRNA hsa_circ_0004123, and verify the copy number of hsa_circ_0004123 in the leukemia cell line. Methods Total RNA was extracted from HMy2.CIR cell line, and reverse transcribed to cDNA. Using cDNA as template, a 236 bp characteristic segment containing the back-spliced junction of hsa_circ_0004123 transcript was cloned and inserted into the pGM-T vector, and then the recombinant plasmid was sequenced. Regarding the gradient diluted recombinant plasmid as standard substance, the standard curve of hsa_circ_0004123 was established by SYBR fluorescent staining RT-qPCR, and then the copy number of hsa_circ_0004123 in leukemia cell lines (HMy2.CIR, Jurkat, Raji, K562) were verified. Results The absolute RT-qPCR method was established that can accurately quantify the copy number of hsa_circ_0004123 with wide linear range (9.63×103–9.63×109) copies/μl, high sensitivity (4528±516) copies/μl, intra-batch coefficient of variation (CV) of 0.30%-1.86%, inter-batch CV of 1.40%-2.30%, and a single peak dissolution curve. The copy number of hsa_circ_0004123 in leukemia cell lines HMy2.CIR, Jurkat, Raji and K562 was detected respectively. The expression of hsa_circ_0004123 was significantly different (P<0.001) in the 4 kinds of leukemia cell lines. Conclusion The absolute RT-qPCR method used to detect the copy number of hsa_circ_0004123 has a wide linear range, high sensitivity and specificity, and good repeatability, and lays a technical foundation for accurate quantification of hsa_circ_0004123 in leukemia.