Total RNA was isolated from chondrocyte cultures with TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. The RNA was reverse transcribed with qScript cDNA Supermix reagents (Quanta BioSciences, Gaithersburg, MD) and amplified at 42 °C for 30 minutes. For real-time reverse transcription–polymerase chain reaction (RT-PCR), the PCR products were detected using RT2 Real-Time SYBR Green reagents (SABiosciences, Frederick, MD). Primer-specific amplification was performed at 60 °C for 30 seconds. However, fluorescence quantification was performed at a higher temperature (72°C). The primers pair sequences for bovine genes, forward and reverse, are as follows; GAPDH, 5′-ATTCTGGCAAAGTGGACATCGTCG-3′, 5′-ATGGCC TTTCCATTGATGACGAGC-3′; ADAMTS4, 5′-TCACTG ACTTCCTAGACAATGG-3′, 5′-ACTGGCGGTCAGCGT CGTAGT-3′; ADAMTS5, 5′-CACCGTGGCTCACGAAA TTG-3′, 5′-GGAGCCGAAATTTTCTTCACAGA-3′. All primers were obtained from Integrated DNA Technologies (Coralville, IA). Thermal cycling and fluorescence detection were performed using the SmartCycler System (Cepheid). Real-time PCR efficiencies and the fold increase in copy numbers of messenger RNA (mRNA) were calculated as described previously.34 (link) The data are presented as mean ± standard deviation (SD) by setting negative control as 1.0, and analyzed by Student’s t test. P values <0.05 were considered significant.
Qscript cdna supermix reagent
Manufactured by Quanta Biosciences
The QScript cDNA SuperMix Reagent is a ready-to-use solution for the reverse transcription of RNA into complementary DNA (cDNA). It contains the necessary components for the reverse transcription reaction, including a reverse transcriptase enzyme, oligo(dT) primers, and dNTPs. The reagent is designed to enable efficient and reliable cDNA synthesis from total RNA or poly(A)+ RNA samples.
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Lab products found in correlation
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Rt2 real time sybr green reagents, by Qiagen (2 mentions)
Smart cycler system, by Cepheid (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Applied biosystems viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Ribozol rna extraction reagent, by Avantor (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Qscript microrna quantification system, by Quanta Biosciences (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cfx connect real time system machine, by Bio-Rad (1 mentions)
Sybr master mix, by Thermo Fisher Scientific (1 mentions)
7 protocols using qscript cdna supermix reagent
1
Gene Expression Profiling of Chondrocytes
2
Quantitative RT-PCR Analysis of Chondrocyte Genes
3
RNA Extraction and Quantification Protocol
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RNA from MCs was extracted using Ribozol RNA Extraction Reagent (Amresco) as per the manufacturer’s recommendation, with 1 μg of RNA reverse transcribed into cDNA using qScript cDNA SuperMix Reagent (Quanta Biosciences). miRNA-enriched cDNA was generated using the qScript microRNA Quantification System (Quanta Biosciences). Quantitative real-time PCR was carried out using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) on the Applied Biosystems ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). mRNA and miRNA expression and fold changes were calculated using the ΔΔCT method, where 18S was used as a control for mRNA and U6 snRNA as a control for miRNA. Primers sequences used in the study are provided in Supplementary table 5 .
4
Quantitative Analysis of Chondrogenic Markers
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Total RNA was extracted from cells using TRIzol (Invitrogen, Shanghai, China), in accordance with the manufacturer's instructions. RNA was then reverse transcribed using qScript cDNA SuperMix reagent (Quanta BioSciences, Beijing, China), and relative gene expression was determined by qRT-PCR and the 2−ΔΔCT method. The primer sequences were as follows: SOX9 (5′-AGCAAGAACAAGCCCCACGTC-3′, 5′CCTGCCCATTCTTCACCGACT-3′); ACAN (5′-CATCTGGAGTTCTTTTTGGGAG-3′, 5′-CAGGTCAGGGATTCTGTGTGTC-3′); COL2A1 (5′-GAAGACACCAAGGACTGCCTG-3′, 5′-GCACCCTTTTCGCCTTTGTCA-3′); PPARγ (5′-TGCAGGAGCAGAGCAAAGAAG-3′, 5′-GAGGCCAGCATGGTGTAGATG-3′); and Runx-2 (5′-TGATGACACTGCCACCTGTG-3′, 5′-ACTCTGGCTTTGGGAAGAGC-3′). Each experiment was repeated in triplicate.
5
Quantitative real-time PCR for gene expression analysis
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RNA was extracted using TRIzol (Invitrogen) and 1 µg was reverse transcribed to cDNA using qScript cDNA SuperMix Reagent (Quanta Biosciences) for quantitative real-time PCR using Power SYBR Green PCR Master Mix (Thermo Fisher) on the Applied Biosystems ViiA 7 Real-Time PCR system. Primers were: TRII F5′-GGTCTATGACGAGCGACGGG-3′, R5′-GCTTCCATTTCCACATCCGAC-3′; TGFβ1 F5′-AAACGGAAGCGCATCGAA-3′, R5′-GGGACRGGCGAGCCTTAGTT-3′, fibronectin F5′-GATGGAATCCGGGAGCTTTT-3′, R5′-TGCAAGGCAACCACACRGAC-3′; collagen Iα1 F5′-CTTCACCTACAGCACCCTTGTG-3′, R5′-GATGACTGTGCTTGCCCCAAGTT-3′; αSMA F5′-GACGCTGAAGTATCCGATAGAAC-3′; R5′-GGCCACACGAAGCTCGTTAT-3′; ALK4; F5′-CTGTTTGATTATCTGAACCG-3′; R5′-ACAACCTTTCGCATCTCCTC-3′; ACRIIA; F5′-GTTGAACCTTGCTATGGTGATAA-3′; R5′-AATCAGTCCTGTCATAGCAGTTG-3′; ACRIIB F5′-CACAAGCCTTCTATTGCCCACAG-3′; R5′-ATFTACCGTCTGGTGCCAAC-3′. Gene expression was calculated using the ∆∆CT method with 18S (F5′-GCCGCTAGAGGTGAAATTCTTG-3′, R5′-CATTCTTGGCAAATGCTTTCG-3′) used as an internal control.
6
RNA Extraction and RT-qPCR Analysis
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RNA from MC was extracted using Ribozol RNA Extraction Reagent (Amresco) as per the manufacturer’s recommendation, with 1 μg of RNA reverse transcribed into cDNA using qScript cDNA SuperMix Reagent (Quanta Biosciences). Quantitative real-time PCR was carried out using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) on the Applied Biosystems ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). mRNA expression and fold changes were calculated using the ΔΔCT method, where 18S was used as the endogenous control. Primer sequences used in the study are provided in Additional file 1 : Table S4.
7
Reverse Transcription and qRT-PCR Protocol
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Here, 1 µg of RNA was reverse transcribed to cDNA using 4 µl of qScript cDNA SuperMix reagent (Quanta Bioscience, 95048–100) in a final reaction volume of 20 µl. PCR cycling conditions were as follows: 5 min incubation at 25 °C, DNA polymerisation at 42 °C for 30 min and enzyme deactivation at 85 °C for 5 min. PCR product was diluted 1:10. For the qRT-PCR experiment, a reaction mixture of 20 µl containing 10 µl of SYBR Master mix (Life Technologies, A25742), 0.5 µM of each reverse and forward primer, 5 µl of cDNA template and 4 µl of RNase-free water was prepared. The list of primers used are shown in Table S1 . Reactions were conducted in duplicate and β-actin was used as a reference gene for CT-value normalisation. Amplification was conducted using the CFX Connect Real-Time System machine (Bio-Rad) and cycling conditions were as follows: a pre-incubation step of 95 °C at 15 min, followed by an amplification step of 95 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s repeated for 44 cycles, and a melting curve analysis from 65 °C to 95 °C in 0.5 °C intervals.
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