Steponeplus q pcr detection system

Quantitative polymerase chain reaction (qPCR) was undertaken based on a previously reported technique [42 (link), 43 (link)]. Briefly, reactions were prepared containing, PrecisionFAST qPCR mastermix (Primer Design, Eastleigh, UK), forward primer, z-tagged reverse primer (Table 1), Uniprimer probe (Intergen Inc., Oxford, UK), molecular biology grade water and sample cDNA and were run on a StepOne Plus qPCR detection system (Life Technologies, Paisley, UK). Reaction conditions were; initial 95° C for 15 minutes followed by 100 cycles of 95° C for 15 seconds, 55° C for 35 seconds and 72° C for 20 seconds. Samples were run simultaneously with a standard of known transcript copy number, allowing calculation of relative transcript expression. Quantification of GAPDH expression was subsequently used to normalize samples.

Real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) was performed using GoTaq^TM 2‐step RT‐qPCR System (Promega) on the StepOnePlus QPCR Detection System (Life Technologies) following the manufacturer's instructions. Glyceraldehyde‐3‐phosphate dehydrogenase (GADPH) mRNA was used to normalize the levels of the circRNA. The sequences of hsa_circ_001888 divergent primers were as follows: Forward: 5′‐ TCCAGTGGCTCCCGTTTC‐3′ and Reverse: 5′‐ CGCCCAGCTTGCACTCA‐3′. The primers were synthesized by BGI Genomics (Guangdong, China). The reaction conditions were as follows: 95°C for 5 min for a not start, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. The C_q values were recorded for both hsa_circ_001888 and GADPH.
All reactions were performed in triplicate. The appearance of a single‐peak in the melt‐curve suggested the specificity of the PCR products. Relative quantification of gene expression was analyzed using ΔC_q method. All results are expressed as the mean ± SD.