Lightcycler 96 real time system

Total RNA was extracted from goat tissue samples or cultured cells using the reagent Trizol (Takara, Dalian, China). The concentration of total RNA measured by NanoDrop2000 (Thermo, San Jose, CA, USA) and the quality was checked by denaturing agarose gel electrophoresis. The cDNA synthesis was performed with reverse transcription kits (Takara, Dalian, China). For quantification of miR-487b-3p expression, miRNA specific complementary DNA was generated using miRNA stem-loop-specific primers (Table 1). The cDNA generated was stored at −20 °C for subsequent usage.
qRT-PCR was performed using TB green premix Ex taq II (Takara, Dalian, China) on a Light Cycler 96 real-time system (Roche, Basel, Switzerland) with a reaction volume of 25 μL. Each sample was carried out in triplicate and repeated for three times at least. The mRNA levels of all coding genes were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal standard. On the other hand, the expression level of miR-487b-3p was quantified with 18S RNA as the reference. The relative gene expression was analyzed using the comparative threshold cycle (CT) method (2^−∆∆Ct). The primers of the target genes are listed in Table 1.

RNA of mouse colons was isolated using the FastPure cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China) and reverse-transcribed into complementary DNA (cDNA) using HiScript III RT SuperMix (Vazyme) according to the manufacturer's instructions. Primers and PCR product for qRT-PCR are listed in Supplementary Table S1. qRT-PCR was carried out using AceQ qPCR SYBR Green Master Mix (Vazyme) by preheating at 95 °C for 5 min followed by 40 cycles at 95 °C for 10s and 60 °C for 30s on the LightCycler 96 Real-Time System (Roche, City, USA).

Total RNA was extracted using the EASYspin Plus Plant RNA Kit (Aidlab, Bejing, China), according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using M-MLV Reverse Transcriptase according to the instructions (Promega, Madison, WI, USA). The real-time qPCR was performed using TransStart Top Green qPCR Supermix (Transgene, Beijing, China) on a Roche LightCycler96 Real-Time System. The 2^−ΔΔCt method was used to evaluate the expression levels. Three independent replications were performed for each experiment. The housekeeping gene AsTUB was used as the internal control, as described by Gao et al. [90 (link)]. All primers were designed using the online tool Primer3Plus (https://www.primer3plus.com/, accessed on 25 September 2022) and are listed in Table S1. Because AsJAZ7 and AsJAZ8 had highly similar sequences, they used the same pair of qPCR primers; AsJAZ4 and AsJAZ5 also used the same pair of primers for their highly similar sequences.

Cellular RNA was isolated with TRIzol reagent (TaKaRa, Kusatsu, Japan). Next, a NanoDrop 2000 spectrophotometer device (Thermo Fisher Scientific, Waltham, MA, USA) was used for detection of the purity and concentration. RNA was transcribed into cDNA using the PrimeScript RT Reagent Kit (Takala, Japan). qRT-PCR was conducted with a standard SYBR Green PCR kit protocol (Roche, Basel, Switzerland) and performed in a LightCycler 96 Real-Time System (Roche, Switzerland). The expression levels of β-actin were used to normalize the results. The primer sequences used are displayed in Table 2.

The primer pairs used for qRT-PCR were designed and synthesized according to the sequence information of BCAS2 and β-actin from NCBI, and the details are shown in Table 1. The β-actin was used as a housekeeping gene for the normalization of BCAS2 mRNA expression. The qRT-PCR was carried out in LightCycler 96 Real-Time System (Roche, Basel, Switzerland) following optimized procedures: one cycle of 95 °C for 3 min; 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s. The amplification reaction system (20 µL) comprised of 0.8 µL of each forward and reverse primer (10 µM), 2 µL of cDNA template (100 ng), 6.4 µL of RNase free ddH₂O, and 10 µL of 2× SYBR Premix Ex Taq (Takara, Dalian, China). The relative expression level of BCAS2 mRNA was evaluated relative to that of β-actin using the 2^−∆∆Ct method.

PrimeScript RT Master Mix (TaKaRa RR036A) was used for RNA reverse transcription. Quantitative PCR was conducted with FastStart Essential DNA Green Master on the Roche LightCycler 96 Real‐time System. The relative expression was normalized to β‐actin using the 2^−ΔΔCq method. The following thermocycling conditions were used: initial denaturation at 95 °C for 5 min and 40 cycles of 10 sec at 95 °C and 30 s at 60 °C. The primers used in this study were shown in Table1:

For the quantitative RT-PCR analysis, total RNA was extracted using the RNA extraction kit, and a reverse transcription kit (Takara, Kusatsu, Japan) was used for the first complementary DNA (cDNA) synthesis. The RT-PCR primers of candidate genes (Table S3) were designed using primer blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast; accessed on 17 May 2022). Diluted cDNA was amplified using TB Green premix EX Taq II. PCR was performed on a LightCycler96 Real-Time System (Roche, Basel, CH). Each sample was performed with three independent biological replicates. The actin gene was used as an internal control [21 (link)].