DRGs were subjected to TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for RNA extraction. The purity and concentration of the extracted RNA samples were determined by spectrophotometric analysis. Reverse transcription was performed using an EasyScript First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China) with 3 micrograms of RNA. Quantitative real-time PCR (qRT-PCR) was used to evaluate the relative mRNA expression levels of mouse Atf3, Sox11, Lin28a, Gap43, Smad1, Lin28b, Jun and Gapdh (Invitrogen), utilizing TransStart Eco Green qPCR Super Mix (TransGen Biotech). The Bio-Rad myiQ2 Sequence Detection System (Bio-Rad, Hercules, CA, USA) was employed in this study. Primers purchased from Invitrogen were utilized as per Supplementary Table S1 instructions while cycle conditions followed the specifications of the primers. Relative gene expression levels were normalized against Gapdh and β-actin and analyzed using the 2−ΔΔct method.
Transstart eco green qpcr super mix
Manufactured by Transgene
Sourced in United States, China
TransStart Eco Green qPCR Super Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) applications. It contains all the necessary components, including a DNA polymerase, buffer, and green fluorescent dye, for the efficient and sensitive detection and quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Easyscript first strand cdna synthesis supermix kit, by Transgene (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Myiq2 sequence detection system, by Bio-Rad (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rna isolation kit, by Takara Bio (1 mentions)
Improm 2 reverse transcription system, by Promega (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Nucleospin mirna kit, by Macherey-Nagel (1 mentions)
Transscript first stand cdna synthesis kit, by Transgene (1 mentions)
6 protocols using transstart eco green qpcr super mix
1
Quantitative Analysis of Neuronal Transcripts
2
Evaluation of Osteogenic Differentiation in Human Mesenchymal Stem Cells
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Total RNA was extracted from human mesenchymal stem cells (h-MSCs) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and purity of RNA samples were assessed by spectrophotometric analysis. Three micrograms of RNA was used to reverse transcription by using an EasyScript First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China). The relative mRNA expression of human P2X7, RUNX2, ALP, OPN, and GAPDH (Invitrogen) was evaluated by quantitative real-time PCR (qRT-PCR) by using the BioRad myiQ2 Sequence Detection System (BioRad, Hercules, CA, USA) and TransStart Eco Green qPCR Super Mix (TransGen Biotech, Beijing, China). Primers were purchased from Invitrogen, and the sequences were listed in Additional file 1 : Table S1. Cycle conditions followed primers’ introduction. Relative gene expressions were normalized with GAPDH and analyzed by the 2−ΔΔCt method.
3
Gene Expression Analysis of Osteogenic Markers
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Total RNA was extracted using TRIzol reagent. The purity and concentration of the RNA samples were determined spectroscopically. First-strand cDNA was synthesized from 3 µg RNA, using an EasyScript First-Strand cDNA synthesis super mix kit (TransGen Biotech Co., Ltd., Beijing, China) and used for qPCR. The expression of runt-related transcription factor 2 (RUNX2), ALP and osteopontin (OPN) was quantified using a Bio-Rad MyiQ2 sequence detection system and TransStart Eco Green qPCR SuperMix (TransGen Biotech Co., Ltd.). The primers were synthesized by Invitrogen, and their sequences are listed in Table I . The reactions were incubated at 95°C for 30 sec, followed by 40 cycles of 94°C for 5 sec and 60°C for 35 sec. The relative expression of gene-specific products was analyzed using the 2−ΔΔCt method and normalized to the corresponding GAPDH values.
4
Quantitative Real-Time PCR for Gene Expression
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Total RNA was extracted using Trizol reagent according to the manufacturer's instructions. RNA was quantified spectroscopically, and 3 μg RNA was reverse transcribed using an EasyScript First‐Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China). Human P2X7, E‐cadherin, fibronectin, vimentin, snail and GAPDH mRNA listed in Supporting Information Table S1 were quantified with quantitative real‐time PCR. Bio‐Rad myiQ2 Sequence Detection System (Bio‐Rad, Hercules, CA) and TransStart Eco Green qPCR Super Mix (TransGen Biotech, Beijing, China) were used according to the manufacturer's protocol. Cycle conditions were as follows: 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 35 s. Relative gene expression was assayed by normalizing with GAPDH using the 2−ΔΔCt method.
5
Quantifying MicroRNA and mRNA Expression
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Micro RNA was extracted by NucleoSpin miRNA kit (Macherey–Nagel, France) and total RNA by an RNA isolation kit (Takara Bio, Japan). A260/A280 absorbance ratio was quantified to assess RNA purity. Micro RNA was reverse-transcribed into cDNA by the ImProm-II Reverse Transcription System (Promega, WI, United States). TransStart Eco Green qPCR SuperMix (TransGen Biotech, Beijing, China) was used in a CFX Connect Real-Time PCR Detection System (Bio-Rad, United States) for PCR reaction. PrimeScript RT Master Mix (Takara Bio) was used to reverse transcribe mRNA into cDNA. The relative expression of miRNA and mRNA was quantified by the 2−∆∆CT method (24 (link)) and normalized to endogenous reference gene U6 and GAPDH, respectively. Primer sequences for this study are shown in Table 1 .
6
Quantitative Real-time PCR Analysis
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