Total RNAs of cells and tissues were extracted by using TRIzol reagent (TaKaRa, Dalian, China). Total RNAs were reverse transcribed using a PrimeScript RT reagent kit with genomic DNA (gDNA) Eraser to remove gDNA (TaKaRa). Quantitative real-time PCR was performed in triplicate using a SYBR Green kit (Genestar, Beijing, China) on a Bio-Rad CF96 system (Bio-Rad, USA). GAPDH was selected as the internal gene. The 2−ΔΔCt method was performed to analyze the relative expression level of quantitative real-time PCR data. Primer sequences for quantitative real-time PCR are listed in Table S3 .
Cf96 system
Manufactured by Bio-Rad
Sourced in China
The Bio-Rad CF96 system is a real-time PCR detection system designed for accurate and reliable quantification of DNA and RNA targets. The system features a compact footprint, a 96-well format, and a temperature range of 4-100°C. It is capable of running various real-time PCR applications, including gene expression analysis, pathogen detection, and genotyping.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Takara Bio (2 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Sybr green kit, by Takara Bio (1 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Sybr green qpcr master mix, by Vazyme (1 mentions)
Hiscript 3 reverse transcriptase kit, by Vazyme (1 mentions)
4 protocols using cf96 system
1
Quantitative Real-Time PCR Analysis
2
Buffalo Tissue RNA Extraction and qPCR
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The total RNA was extracted from buffalo tissues and myoblasts using Trizol reagent (TaKaRa, Dalian, China). PrimeScript™ RT kit (TaKaRa, Dalian, China) with gDNA eraser was used to reverse transcribe the total RNA while removing genomic DNA. The SYBR Green kit (TaKaRa, Dalian, China) was used to perform qPCR in triplicate on a Bio-Rad CF96 system (Bio-Rad, USA), and the data was normalized using β-actin. The primers used are listed in Supplementary Table S1 .
3
Validating RNA-Seq Findings with qRT-PCR
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Results of RNA-Seq analysis were validated using qRT-PCR. Total RNA (2 μg) prepared for RNA-Seq was used for the synthesis of first-strand cDNA. Subsequently, qRT-PCR was conducted with TransStart Top Green qPCR SuperMix (TransGen Bio-tech, Beijing, China) using a CF × 96 system (Bio-Rad, Hercules, CA, USA). A total of eight differentially expressed genes, including PRRs (CTL4, CTL7), AMPs (gloverin, cecropin 3), SPs (SP4, azurocidin-like SP), and mucins (mucin protein, mucin 4), were selected for qRT-PCR analyses. The expression level of each gene was normalized relative to that of the reference gene β-actin using the 2-ΔCT method (ΔCT = CT[test gene] - CT[β-actin]). Gene-specific primers used for qRT-PCR analysis are listed in Additional file 6 : Table S4.
4
Bioinformatic Prediction of miRNA-mRNA Interactions
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Based on the binding sites of miRNAs detected in mRNA sequences, the RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/ ) and MiRanda (http://cbio.mskcc.org/microrna_data/miRanda-aug2010.tar.gz ) software were used to predict the interactions of miRNAs and mRNAs.
cDNA Synthesis, and Real-time qPCR.
Total RNA was reversely transcribed using a Hiscript III Reverse Transcriptase Kit (Vazyme, Nanjing, China). Real-time quantitative PCR (qPCR) was performed in triplicate using qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) on a Bio-Rad CF96 system (Bio-Rad, United States), and U6 and GAPDH were used for normalization of the data.
cDNA Synthesis, and Real-time qPCR.
Total RNA was reversely transcribed using a Hiscript III Reverse Transcriptase Kit (Vazyme, Nanjing, China). Real-time quantitative PCR (qPCR) was performed in triplicate using qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) on a Bio-Rad CF96 system (Bio-Rad, United States), and U6 and GAPDH were used for normalization of the data.
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