Sybr green quantitative polymerase chain reaction

Total cellular RNA was isolated using an RNeasy Mini Kit (Qiagen, Venlo, Limburg, the Netherlands). RNA samples were treated with DNA‐free DNase I (Ambion, Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions and were reverse transcribed into complementary DNA (cDNA) using a high‐capacity cDNA reverse‐transcription (RT) kit (Applied Biosystems, Life Technologies, Carlsbad, CA) as per the manufacturer’s instructions. cDNAs were further amplified using SYBR green quantitative polymerase chain reaction (PCR) master mix (Thermo Fischer Scientific), according to the manufacturer’s protocol, for 40 cycles on a ViiA 7 Real‐Time PCR machine (Applied Biosciences). Transcripts were amplified using gene‐specific primers (summarized in Supporting Table S1). A reverse‐transcriptase negative blank of each sample and a no template blank served as negative controls. Gene expression was normalized to the housekeeping gene 18S ribosomal RNA, which was used as an internal standard.

Total cellular RNA was extracted using TRIzol (Invitrogen) and cDNA was synthesized with 2 µg of total RNA and TOPscript RT DryMIX (Enzynomics Inc.) according to the manufacturer’s instructions. Amplification was carried out using SYBR Green quantitative polymerase chain reaction (qPCR) master mix (Thermo Fisher Scientific). The PCR reactions were performed for 40 cycles at 95 °C for 15 seconds, 60 °C for 1 minute and 72 °C for 1 minute. The primer sequences were as follows: MRPL14-F: 5´-GAA GAA AAA GGC GCT CAT TG-3´/MRPL14-R: GAG GAC CAC GTT GTT GGA GT-3´; GAPDH-F: 5´-ACC CAG AAG ACT GTG GAT GG-3´/GAPDH-R: 5´-TTC TAG ACG GCA GGT CAG GT-3´. The cycle threshold (Ct) values provided from real-time PCR instrumentation were imported into Microsoft Excel. We used the 2^−∆∆Ct model for relative quantification of real-time qPCR fold changes.