The ileal mucosa RNA samples were extracted via the Trizol reagent (Tiangen Biochemical Technology Co., Ltd., Beijing, China), which was subsequently assessed for purity and concentration at 260/280 nm. Moloney murine leukemia virus reverse transcriptase (Accurate Bio-Medical Technology Co., Ltd., Hunan, China) was used to reverse-transcribe 1 g of RNA into cDNA. Each RT-qPCR reaction included nuclease-free water, gene-specific forward and reverse primers of each gene, cDNA, and TB Green Premix Ex Taq mix (Takara Biotechnology Co., Ltd., Dalian, China). The primers for RT-qPCR analysis were designed based on sequences retrieved from the GenBank database (Table S2 ). A denaturation stepped at 95°C for 30 s was followed by 40 cycles at 95°C for 5 s and 60°C for 30 s during the RT-qPCR process. The relative mRNA levels of the target genes normalized to β-actin were analyzed using the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)).
Tb green premix ex taq mix
Manufactured by Takara Bio
Sourced in Switzerland
TB Green Premix Ex Taq mix is a ready-to-use solution for real-time PCR amplification and detection. It contains TB Green Dye, Taq DNA polymerase, dNTPs, and buffer components optimized for efficient and sensitive real-time PCR performance.
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5 protocols using tb green premix ex taq mix
1
RNA Extraction and RT-qPCR Analysis Protocol
2
Quantitative RT-PCR Analysis of Mouse Genes
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Total RNA was isolated using TRIzol reagent (Invitrogen, USA) and converted to complementary DNA using the PrimeScript RT reagent kit (RR037A; Takara Biomedical Technology, Beijing, China). PCR was performed using TB Green Premix Ex Taq mix (RR820A, Takara Biomedical Technology) and a CFX96 PCR thermocycler (Bio-Rad, USA). Mouse primer sequences for the target genes are presented in Supplementary Table 1. Gene expression was normalized to the expression of β-actin.
3
RNA Extraction and cDNA Synthesis
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Total RNA was extracted by Trizol reagent (TaKaRa). A total of 1 μg total RNA was reverse transcribed to cDNA by PrimeScript RT reagent Kit (TakaRa). Oligo dT and random primer mix were used for mRNA reverse transcription, while gene-specific reverse transcription primers were used for miR and U6 reverse transcription. The cDNAs were quantified by qPCR with TB Green Premix Ex Taq mix (TakaRa). The primer sequence were shown in Supplementary Table 1 .
4
Quantitative Analysis of Liver RNA
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Total RNA was isolated from mice liver sample using a spin column animal total RNA puri cation kit (Sangon biotech, Shanghai, China) in accordance with the manufacturer's instructions. M-MuLV rststrand cDNA synthesis kit was used for reverse transcription into cDNA with total RNA as template.
MiRNA was isolated with a miRNeasy Mini kit and reverse transcription was performed using the Tailadded reverse transcription kit (Sangon biotech, Shanghai, China). Quantitative PCR was performed on a LightCycler480 system (Roche, Switzerland) using TB Green premix Ex Taq mix (TaKaRa, Dalian, China), gene speci c primers were listed in Table 1, β-actin and U6 small nuclear RNA (snRNA) was used as an internal control.
MiRNA was isolated with a miRNeasy Mini kit and reverse transcription was performed using the Tailadded reverse transcription kit (Sangon biotech, Shanghai, China). Quantitative PCR was performed on a LightCycler480 system (Roche, Switzerland) using TB Green premix Ex Taq mix (TaKaRa, Dalian, China), gene speci c primers were listed in Table 1, β-actin and U6 small nuclear RNA (snRNA) was used as an internal control.
5
RNA Extraction and qPCR Analysis
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The RNA was extracted by the Trizol method [27] , and the air-dried RNA was precipitated for 5-10 min after discarding the supernatant, and the precipitate was dissolved in 20 μl of DEPC water. Take 2μl of the dissolved RNA and measure the OD260, OD280 , and OD260/OD280 values by micro spectrophotometer (Allsheng Instruments Co., Ltd, Hangzhou, China) to calculate the purity and concentration of RNA. The concentration of sample RNA was calculated according to the following formula based on the absorbance value: Total RNA concentration ( μg/μl ) = OD260×40×10 -3 . Quantitative PCR was performed on a LightCycler480 system (Roche, Switzerland) using TB Green premix Ex Taq mix (TaKaRa, Dalian, China). mRNA expression was calculated by the 2^ △△CT method. Gene-speci c primers were listed in Table2 β-actin was used as an internal control.
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