Total RNA content was extracted from the tissues using RNA extraction kits (D203‐01, GenStar Biosolutions,). Reverse transcription procedures were performed with the help of PrimeScript RT reagent kits (RR047A, Takara Holdings Inc.,). The primers were designed and synthesized by Takara Holdings Inc. (listed in Table S1 ). Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) was carried out with RT‐qPCR kits (Q511‐02, Vazyme Biotech,) on a Bio‐Rad's CFX96 real‐time system. The relative expression of each target gene was subsequently calculated based on the 2−ΔΔCt method, with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) serving as the loading control.
Rt qpcr kit
Manufactured by Vazyme
Sourced in China
The RT-qPCR kit is a laboratory tool designed for reverse transcription and quantitative real-time PCR analysis. Its core function is to enable the detection and quantification of specific RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
First strand cdna synthesis kit, by Beyotime (4 mentions)
Cfx96, by Bio-Rad (2 mentions)
Cfx96 real time pcr system, by Bio-Rad (2 mentions)
Real time quantitative pcr instrument cfx96, by Bio-Rad (2 mentions)
Primescript rt kit, by Takara Bio (2 mentions)
Cfx96 real time pcr instrument, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Merck Group (2 mentions)
Cdna synthesis kit, by Beyotime (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Mirna first strand cdna synthesis kit, by Sangon (1 mentions)
Cdna reverse transcription kit, by Sangon (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
Reverse transcription kit, by Vazyme (1 mentions)
Primescript mirna rt kit, by Takara Bio (1 mentions)
Mirna first strand cdna synthesis, by Sangon (1 mentions)
Nanodrop lite uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
18 protocols using rt qpcr kit
1
RNA Extraction and RT-qPCR Analysis
2
Quantitative RT-PCR for Gene Expression
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Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) and then reversely transcribed into cDNA using the cDNA Synthesis Kit (Beyotime, Shanghai, China). Synthesized cDNA was subsequently subjected to RT-qPCR utilizing RT-qPCR kits (Q511–02, Vazyme Biotech, Nanjing, China) and CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA), with GAPDH as an internal control. Further, the 2−ΔΔCt method was used for relative quantification. Primers were commercially designed (Sangon Biotech, Shanghai, China), as listed in Supplementary Table S1.
3
RNA Extraction and RT-qPCR Analysis Protocol
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Total RNA content was extracted from tissues or cells using Trizol kits (16096020, Thermo Fisher Scientific, Waltham, MA, United States). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using RT-qPCR kits (Q511-02, Vazyme Biotech, Nanjing, Jiangsu, China) according to the instruction manual. The primer sequences with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene are shown in Table 1 , which were designed and provided by Sangon Biotech (Shanghai, China).
4
RNA Extraction, cDNA Synthesis, and RT-qPCR Quantification
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Total RNA was extracted using Trizol (16096020). mRNA was prepared using the cDNA first-strand synthesis kit (D7168L, Beyotime), and miRNA was prepared using the miRNA first-strand cDNA synthesis kit with poly(A) tailing (B532451, Shanghai Sangon Biotech). cDNA synthesis was performed following the related instructions.
According to the kit instructions, the RT-qPCR kit (Q511-02, Nanjing Vazyme Biotech) was selected for experiments.
In this experiment, MALAT1 was normalized to β-actin, while miR-382-3p was normalized to U6. Shanghai Sangon Biotech provided the primer sequences (Additional file3 : Table S4). The 2−ΔΔCt method was used to calculate the fold change in the expression of the target gene between the experimental and control groups, where ΔCt represents Ct (target gene) − Ct (internal reference), and ΔΔCt = ΔCt experimental group − ΔCt control group.
According to the kit instructions, the RT-qPCR kit (Q511-02, Nanjing Vazyme Biotech) was selected for experiments.
In this experiment, MALAT1 was normalized to β-actin, while miR-382-3p was normalized to U6. Shanghai Sangon Biotech provided the primer sequences (Additional file
5
Quantitative Analysis of Smurf2 and ALK5 mRNA
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Total RNA was extracted with the TRIzol reagent (16096020, Thermo Fisher Scientific), and the cDNA of mRNA was synthesized using the first-strand synthesis kit (D7168L, Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was carried out using a RT-qPCR kit (Q511-02, Vazyme Biotech, Nanjing, China) according to the instructions. PCR amplification was carried out with Bio-rad real-time quantitative PCR instrument CFX96. β-actin served as the internal reference for Smurf2 and ALK5. All primer sequences were designed and provided by Sangon Biotech. The primer sequences are shown in Table 1 . The relative mRNA expression was measured using the 2-ΔΔCT method (Zhao et al., 2018 (link); Wan et al., 2019 (link)).
6
Quantitative Analysis of VWF and p38 MAPK
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TRIzol reagent (16096020, Thermo Fisher Scientific, New York, USA) was used to extract total RNA. For mRNA detection, reverse transcription kit (RR047A, Takara Bio Inc., Otsu, Shiga, Japan) was used to perform reverse transcription to obtain cDNA. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed according to the instructions of RT-qPCR kit (Q511-02, Vazyme Biotech, Nanjing China). PCR amplification was performed using the Bio-rad real-time quantitative PCR instrument (CFX96). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference of VWF and p38 MAPK. The primer sequence and plasmid construction were completed by Sangon (Additional file 1 : Table S3). The 2−ΔΔCt method was used to quantify the relative expression of target genes.
7
Quantitative Analysis of MALAT1 and ZFP36
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Total RNAs were extracted from tissues or cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). A NanoDrop spectrophotometer (Thermo, St Louis, MO, USA) was used to analyze the amount of RNAs. The cDNA was obtained as per the instructions of mRNA Reverse Transcription Kit (B532451, Sangon Biotech) and cDNA Reverse Transcription Kit (B532445, Sangon Biotech), followed by amplification of gene fragments. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed with a RT-qPCR kit (Q511-02, Vazyme Biotech, Nanjing, China) used conforming to the contained instructions. With 2 μL of cDNA as the template, 0.2 μL of forward and reverse primers were mixed with 10 μL of RT-qPCR Mix and diluted up to 20 μL with RNAase-free water. PCR amplification was performed on a Bio-rad real-time qPCR instrument CFX96. MALAT1 and ZFP36 expression levels were normalized to β-actin and the primer sequences are shown in Supplementary Table 1
8
RNA Extraction and RT-qPCR Analysis
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RNA was extracted using TRIzol reagent (#R401-01, Vazyme, China). RT-qPCR was performed using a reverse transcription kit (#R323-01, Vazyme, China) and RT-qPCR kit (#Q111-02, Vazyme, China) according to the manufacturer’s instructions. Relative gene expression levels were determined using the 2−ΔCt method after normalizing to GAPDH or β-Actin levels. The sequences of primers are listed in Supplementary Table S2 .
9
Quantifying Gene and miRNA Expression
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The total RNA content of cells was extracted with Trizol (16096020, Thermo Fisher Scientific, Waltham, MA, USA) and reversely transcribed into complementary DNA (cDNA) using PrimeScript RT kit (Takara Biotechnology, Dalian, China) and PrimeScript miRNA RT kit (Takara Biotechnology). RT-qPCR was assayed with a RT-qPCR kit (Q511-02, Vazyme Biotech, Nanjing, China) by following the instructions provided. With 2 μL cDNA as templates, the forward and reverse primers (each 0.2 μL) were mixed with 10 μL RT-qPCR and supplemented with RNAase-free water to 20 μL. PCR amplification was carried out with Bio-rad real-time qPCR instrument (CFX96, Bio-Rad). MALAT1 was normalized to GAPDH level, while miR-144 was normalized to U6. The primer sequences were designed and provided by Sangon Biotech. The primer sequences are shown in Additional file 4 : Table S2. The gene expression was measured using the 2-ΔΔCt method with the formula as follows: ΔΔCt = ΔCtexperimental group−ΔCtcontrol group, where ΔCt = Ct(target gene)−Ct(internal reference).
10
Quantification of miR-132 and Acvr2b via RT-qPCR
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The total tissue or RNA was extracted using Trizol (16096020; Thermo Fisher Scientific). The miRs were synthesized using a miRNA first-strand cDNA (tailing method) kit (B532451; Sangon), whereas mRNA was synthesized using cDNA first-strand synthesis kit (D7168L; Beyotime) in accordance with the manufacturer’s instructions. RT-qPCR experiments were performed using an RT-qPCR kit (Q511-02; Vazyme Biotech Co., Ltd., Nanjing, China) in line with manufacturer’s protocol. PCR amplification was performed using a Bio-Rad real-time PCR instrument CFX96. U6 was employed as a miR-132 internal reference while GAPDH was regarded as an internal reference for Acvr2b. The U6 primers and universal downstream primers were provided by miR reverse transcription kit. The primer sequences of miR-132, Acvr2b, and GAPDH were designed and provided by Sangon. The primer sequences are depicted in Table 1 . ΔΔCt = ΔCt experimental group −ΔCt control group, where ΔCt = Ct (target gene) − Ct (internal reference).
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