RNA samples of primary fibroblasts and cybrids were extracted by Tissue/Cell RNA Rapid Extraction Kit (Aidlab, China) according to the manufacturer’s instruction, and the cDNA was synthesized using 1 μg RNA with TRUE script One Step RT-PCR Kit (Aidlab, China). The relative expressions of mitochondrial H strand, as well as 11 genes (NRF-1, PPARA, TFAM, TFB1M, TFB2M, PPARGC1A, ER1, ER2, ESRRA, ESRRG, and FSHR) were measured by RT-qPCR with specific primers (Supplementary Table 1 ). GAPDH was used as the internal control (Li et al., 2016b (link)). The baseline adjustment method of the qPCR software (ANALYTIKJENA, Germany) was used to determine the Ct of each reaction. The amplification efficiencies were close to 100%, and all samples were amplified in triplicate. For data analysis, 2–ΔΔct method was used to calculate the relative level of samples.
Qpcr software
Manufactured by Analytik Jena
Sourced in Germany
The QPCR software is a tool designed for the analysis and interpretation of quantitative real-time PCR (qPCR) data. It provides a user-friendly interface for data management, analysis, and visualization. The software supports various qPCR applications and enables researchers to efficiently process and evaluate their experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Qtower 3g cycler, by Analytik Jena (3 mentions)
Transstart top green qpcr supermix, by Transgene (3 mentions)
Tissue cell rna rapid extraction kit, by Aidlab (1 mentions)
True script one step rt pcr kit, by Aidlab (1 mentions)
Plant rneasy extraction kit, by BioTeke (1 mentions)
Easyscript first strand cdna synthesis supermix, by Transgene (1 mentions)
Transscript first strand cdna synthesis supermix, by Transgene (1 mentions)
4 protocols using qpcr software
1
Quantifying Mitochondrial Gene Expression
2
Quantifying Plant Gene Expression
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RNA extraction, cDNA synthesis and RT–qPCR were performed as previously described (Liu et al., 2018 ) with a few modifications. Briefly, total RNA was extracted using the Plant RNeasy Extraction Kit (BioTeKe, Beijing, China, Cat. No. RP3302). First‐strand cDNA synthesis was conducted using EasyScript® First‐Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China, Cat. No. AE301‐02). Real‐time RT–PCR (RT–qPCR) was performed using TransStart® Top Green qPCR SuperMix (TransGen Biotech, Beijing, China, Cat. No. AQ131‐04). A qTOWER 3G Cycler and qPCR software (Analytik Jena, Germany) were used to perform the experiment and analyse the data. The 2−ΔΔCt method was used to determine the relative abundance of transcripts (Livak and Schmittgen 2001). Ubiquitin (UBQ, FG065618) and tubulin (TU, FG067376) were used as internal controls (Lei et al., 2020 ; Liu et al., 2018 ).
3
Quantitative Real-Time PCR Protocol
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Quantitative RT-PCR reactions were conducted in 96-well plates with a qTOWER 3G Cycler and qPCR software (Analytik Jena, Jena Germany) using the TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). The 20 µL reaction mix contained 10 µL TransStart Top Green qPCR SuperMix, 7 µL nuclease-free water, 1 µL diluted cDNA, and 1 µL of each specific primer (final concentration 10 µM). PCR conditions were 94 • C for 30 s, followed by 45 cycles of 95 • C for 5 s, 59 • C for 15 s, and 72 • C for 10 s. Three technical replicates were performed for each sample to ensure the accuracy of the results. All primers used in the study are listed in Table 2.
4
Quantitative RT-PCR Workflow for Gene Expression
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Highly purified RNA was extracted using the CTAB method [30] (link). The TransScript ® First-Strand cDNA Synthesis SuperMix (TransGen) was used to obtain the cDNA, with the following reaction system: 500 ng of total RNA, 1 µL of anchored oligo(dT) 18 , 10 µL of 2 × ES reaction mix, 1 µL of EasyScript ® RT/RI enzyme mix and appropriate ddH 2 O to make the total volume to 20 µL. The cDNA was diluted 10-fold (cDNA:nuclease free water = 1:10) for further qRT-PCR analysis. Gene-specific primers were designed using Primer Premier 6.25, and NCBI primer-BLAST was used to check their specificity. TransStart ® Top Green qPCR Super Mix (TransGen Biotech, Beijing, China) was employed to carry out the qRT-PCR, with the following reaction system: 10 µL of 2 × TransStart ® Top Green qPCR Super Mix, 7 µL of double-distilled H 2 O, 1 µL of diluted template, and 2 µL of forward primer and reverse primer. qTOWER 3G Cycler and qPCR software (Analytik Jena, Germany) were used as a work program and the 2 -∆∆CT method [31] (link) was used to perform the relative gene expression level analysis. The PtrActin was used as the internal reference gene. Triplicate independent technical and biological replications were used in the analysis. The primers for qRT-PCR are listed in Table S1.
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