Rumen Epithelium SCFA Transporter Expression

Total RNA was extracted from 50 mg of rumen epithelial tissue using a FastPure cell/tissue total RNA isolation kit V2 (RC112; Vazyme, Nanjing, China) to detect the expression level of the SCFA transporter genes. The purity and concentration of total RNA were detected using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The reaction system for reverse transcription was adding 0.4 μL of RT mix, 1,000 ng of total RNA, and RNase-free ddH₂O to make the volume of 20 μL, using a FastKing gDNA Dispelling RT Super Mix (Tiangen, Beijing, China). The reaction procedure was set as 42°C for 15 min and 95°C for 3 min.
The gDNA were used as the templates for RT-PCR by using a 2×TSINGKE Master qPCR mix (SYBR green I, TSE201; Tsingke, Beijing, China) with an ABI7500 (Thermo Fisher) sequence detector. The reaction system for the PCR was as follows: qPCR mix (10 μL); forward/reverse primer, 0.8 μL (10 μM); 50× ROX reference dye, 0.4 μL; and ddH₂O, up to 20 μL. The PCR procedure followed a previously described protocol (43 (link)). A standard curve method and QuantStudio 7 Flex real-time PCR software (Applied Biosystems, Pleasanton, CA) were used for data analysis according to the 2^−ΔΔCT method (51 (link)). The specific primers for genes SLC16A1, SLC16A3, SLC9A1, SLC9A3, SLC4A2, ATP6V1E1, and ATP1A1 are shown in Table S6 in the supplemental material.