Total RNA was isolated using RNA pure Tissue & Cell Kit (#CW0560, Cwbiotech; Beijing, China), and cDNA was synthesized using ReverTra Ace qPCR RT Kit (#FSQ-101, Toyobo; Osaka, Japan), according to the manufacturer’s protocol. Amplification and detection were performed with Power SYBR Green Master Mix (#CW0659S, Cwbiotech) and Gentier 48R System (Tianlong Technology, Xi’an, China) using the listed primers (Table S2 ). The observed copy numbers of target genes were normalized to internal GAPDH expression and quantified by 2−ΔΔCt method [15 (link), 16 (link)].
Power sybr green master mix
Manufactured by CWBIO
Sourced in China
Power SYBR Green Master Mix is a ready-to-use solution for real-time PCR amplification and detection using the SYBR Green I dye. It contains all the necessary components, including a hot-start DNA polymerase, SYBR Green I dye, dNTPs, and buffer.
Automatically generated - may contain errors
Lab products found in correlation
Rnapure tissue cell kit, by CWBIO (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Revertra ace qrt pcr rt kit, by Toyobo (1 mentions)
Applied biosystems viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
M mlv, by Vazyme (1 mentions)
Viia7, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Pikoreal 96 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Dnase dna free water, by CWBIO (1 mentions)
4 protocols using power sybr green master mix
1
Quantitative Gene Expression Analysis
2
Quantitative Analysis of Epithelial-Mesenchymal Regulators
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Total RNA was isolated using an RNA pure Tissue & Cell Kit (CW Biotech, Beijing, China), and cDNA was synthesized using a ReverTra Ace qRT-PCR RT Kit (Toyobo, Osaka, Japan) and miRNA cDNA Synthesis Kit, with Poly(A) (Applied Biological Materials, Richmond, BC, Canada) as per the manufacturer’s protocol. Amplification and detection were performed with Power SYBR Green Master Mix (Cwbiotech) and Gentier 48R System (Tianlong Technology, Xi’an, China). The primers are listed in Supplementary Table S2 . For quantitative analysis, the relative mRNA levels of β-catenin, SMAD4, ZEB2, KLF4 and PTEN were normalized to GAPDH, and the relative miRNA level of miR92a was normalized to U6. The primers are listed in Supplementary Table S2 .
3
Cerebellum RNA Extraction and qRT-PCR Analysis
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TRIzol reagent (Invitrogen, 15596–026) was used to extract RNA from the cerebellum of mice. The total RNA concentration and purity were measured by the Nanodrop 1000™ (Thermofisher Scientific) and cDNA was synthesized using M‐MLV (Vazyme, R223‐01), according to the manufacturer's instructions. Quantitative real‐time PCR (RT‐PCR) was performed using Power SYBR Green Master Mix (Cwbio, cw2601) on the Applied Biosystems ViiA 7 real‐time PCR system (Thermo Fisher Scientific), and analyzed using Viia 7 software (Thermo Fisher Scientific). The primers used for RT‐PCR analysis in this study were provided in Table 3 .
4
Milk mtDNA Quantification Using Real-Time PCR
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Ten-fold serially diluted mtDNA extracts (ranging from 100 to 0.01 ng) from raw, pasteurized, retorted, and UHT milk were analyzed to determine the sensitivity of the assay based on primer pair 12S rRNA as described above. The standard curves were obtained by plotting the cycle threshold (Ct) values of realtime PCR analysis against the logarithm of the initial mtDNA amount. Real-time PCR was performed under standard 3-step thermal cycling conditions: 95°C for 10 min followed by 40 cycles of 94°C for 30 s, 60°C annealing for 60 s, and 72°C extension for 60 s using a PIKO REAL 96 real-time PCR System (Thermo Fisher Scientific Inc.). Reactions were carried out with 5 μL of Power SYBR Green Master Mix (CWBIO), 0.4 mM assay-specific primers, 1 μL of normalized template (corresponding to absolute mtDNA amounts of 100, 10, 1, 0.1, and 0.01 ng, respectively), and made up to 10 μL with DNase-/DNA-free water (CWBIO).
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