Abi quantstudio instrument

To validate the relative expressions patterns obtained by RNA-seq, qRT-PCR was performed for four individuals with extremely high IMF and four individuals with extremely low IMF contents. Total RNA was isolated using TRIzol reagent (Invitrogen, United States) and then reverse-transcribed with ABScript III RT Master Mix for qPCR with gDNA Remover (RK20429) (ABclonal, China) following the manufacturer’s instructions. cDNA was quantified using 2X Universal SYBR Green Fast qPCR Mix (RK21203) (ABclonal, China) on an ABI QuantStudio instrument (Applied Biosystems, United States). The amplification regime was conducted with 40 cycles of 95°C for 15 s, 61°C for 15 s, and 72°C for 20 s. The primers used in this study are shown in Table 1. Each gene was normalized to the housekeeping gene β-actin. Three biological replicates were used.

Total RNA was isolated using RNAiso Plus (#9109, Takara Bio, Shiga, Japan), and 1 μg of the obtained total RNA was subjected to reverse transcription to generate cDNA. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted using a TOPreal qPCR 2X PreMIX (#RT500M, Enzynomics, Daejeon, Republic of Korea) and an ABI QuantStudio instrument (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The accuracy of each PCR amplicon was confirmed by obtaining a melting curve. The relative levels of each mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculated using the 2^−∆∆Ct method. The primer sequences utilized for qRT-PCR can be found in Table S1.