TRIzol reagent (Invitrogen, USA) was used to extract the total RNA of cells according to the manufacturer’s instructions. cDNA was synthesized from total RNA (2 μg) by reverse transcription (Thermo Fisher Scientific, USA) according to the instructions provided by the manufacturer. RT-PCR was performed using SYBR QPCR Master Mix (Vazyme) with certain sense (S) and antisense (AS) primers, and realplex2 qPCR system (Eppendorf, Germany). The information on RT-PCR primer pairs was presented in Supplemental information Table S2 .
Realplex2 qpcr system
Manufactured by Eppendorf
Sourced in Germany
The Realplex2 qPCR system is a real-time PCR thermal cycler designed for quantitative gene expression analysis. It features a high-performance optical detection system and can accommodate a range of sample volumes and formats. The Realplex2 enables users to perform accurate and reliable quantitative PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green, by Toyobo (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr qpcr master mix, by Vazyme (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Custom oligonucleotide primers, by Integrated DNA Technologies (1 mentions)
Perfecta sybr green supermix, by Quanta Biosciences (1 mentions)
Qscript supermix, by Quanta Biosciences (1 mentions)
4 protocols using realplex2 qpcr system
1
RNA Extraction and Quantification Protocol
2
Quantitative PCR Analysis of Cell Cycle Genes
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The total RNA was isolated by Total RNA Kit (Omega, USA) according to the manufacturer’s protocols. The cDNAs were synthesized with M-MLV Reverse Transcriptase (Promega, USA) from total RNA samples. qPCR was performed with SYBR Green (Toyobo, Japan) with following sense (S) and antisense (AS) primers: hFOXM1-S, 5′-GCT TGC CAG AGT CCT TTT TGC-3′ and hFOXM1-AS, 5′-CCA CCT GAG TTC TCG TCA ATG C-3′; hCdc25B-S, 5′-AGT CCT GAC CGG AAG ATG GA-3′ and hCdc25B-AS, 5′-GAT GTT GCT GAA CTT GCC CG-3′; hCyclinB1-S, 5′-GGT CTG GGT CGG CCT CTA CCT-3′ and hCyclinB1-AS, 5′-AGC CAG GTG CTG CAT AAC TGG AA-3′; hGAPDH-S, 5′-GGA GCG AGA TCC CTC CAA AAT-3′ and hGAPDH-AS, 5′-GGC TGT TGT CAT ACT TCT CAT GG-3′. The qPCR was performed in the realplex2 qPCR system (Eppendorf, Germany).
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted using Trizol reagent (Omega) according to the manufacturer's instructions. Total RNA (2.0 μg) was reverse transcribed into 20 μl cDNA by RevertAid First Strand Kit (Promega). The qPCR was performed with SYBR Green (Toyobo) with certain sense (S) and antisense (AS) primers. The detail information of primers was described in Supplementary Materials and Methods. The qPCR was performed in the realplex2 qPCR system (Eppendorf).
4
Quantifying Gene Expression in Cell Cultures
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Total RNA was isolated from cultures using TriZol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturers recommended instructions. cDNA was reverse transcribed from 1 µg total RNA using qScript SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) according to manufacturers instructions. qPCR was carried out using a 5 ng equivalent of cDNA in a 1X reaction of PerfeCTa SYBR Green SuperMix (Quanta Biosciences, Beverly, MA, USA) and 250 nM each (forward and reverse) custom oligonucleotide primers (Integrated DNA Technologies, Coralville, IA, USA) generated by using PrimerBlast (National Center for Biotechnology Information, Bethesda, MD, USA). Reactions were carried out on an Eppendorf RealPlex2 qPCR system, and fold changes in gene expression were calculated using the ΔΔCT method using species-specific GAPDH primers. Primer sequences are listed in Table S1 . Human collagen 1 and α-SMA expression levels in the 2D FibCon condition were used as the reference group for fibroblast gene expression, and mouse myogenin gene expression levels of 2D MyoCon were used as a reference for myoblasts. N = 5 for all groups.
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