Quantstudio 7 flex real time pcr software

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.

Extracted gDNA samples were used as templates for quantitative real-time PCR (RT-qPCR) using the 2× TSINGKE Master qPCR Mix (SYBR Green I) (TSE201, Tsingke, Beijing, China) in an ABI7500 (Thermo Fisher Scientific, Waltham, MA, USA) sequence detector. Three replicates per sample in each pair of primers were examined. PCR mixtures included 10 μL 2 × Mix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 1 μL template gDNA (80 ng/μL), and 8.2 μL distilled H₂O. The PCR cycling conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s, ultimately tested at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The standard curve method and QuantStudio™ 7 Flex Real-Time PCR Software (Applied Biosystems, Foster, CA, USA) were used for data analysis. Relative abundance of gut microbes was determined using the 2^−ΔΔCt method [60 (link)], where the 16S rRNA gene amplified by the total bacterial primer set was used as an internal reference [61 (link),62 (link)]. Thus, the standard (NLD) group was considered as a control, and Fold Changes in abundance were expressed relative to the standard (NLD) group. Primer sets for quantification of the Bacteroidetes phylum, Firmicutes phylum, Clostridiales order, Lachnospiraceae family, and Ruminococcaceae family were designed and synthesized by Sangon Biotech Co., Ltd., Shanghai, China, (Table 1).