Total RNA was extracted from cells treated with BA at indicated concentration or was transfected with siRNA or vectors by using Trizol reagent (Invitrogen). cDNA reverse transcription and qRT-PCR were performed as the instruction indication of Reverse Transcription PrimeScript 1st Stand cDNA Synthesis kit (TaKaRa, Otsu, Japan) and quantitative PCR reagents SYBR PremixEx TaqTM (TaKaRa). The relative expression of genes was calculated with the 2−ΔΔCt method. The sequences of the primers used are presented in Supplementary Table 2 .
Reverse transcription primescript 1st stand cdna synthesis kit
Manufactured by Takara Bio
Sourced in Japan
The Reverse Transcription PrimeScript 1st Stand cDNA Synthesis kit is a laboratory tool used for the conversion of RNA into complementary DNA (cDNA). It enables the synthesis of first-strand cDNA from various types of RNA templates, including mRNA, total RNA, and small RNA.
Automatically generated - may contain errors
6 protocols using reverse transcription primescript 1st stand cdna synthesis kit
1
Quantitative RT-PCR for Gene Expression
2
Quantitative RT-PCR Analysis of DJ-1 Expression
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Total intracellular RNA was extracted by Trizol reagent (Invitrogen), and reverse transcription was performed using Reverse Transcription PrimeScript 1st Stand cDNA Synthesis kit (TaKaRa, Otsu, Japan). Following the instructions of manufacture, qRT-PCRs were performed using the quantitative PCR reagents SYBR PremixEx TaqTM (TaKaRa). The fold-changes were analyzed using the 2−ΔΔCt method, which uses the levels of GAPDH quantified with target genes to act as an internal control. The qRT-PCR primers for DJ-1 were as follows: Forward primer: 5′-GTGCAGTGTAGCCGTGATGT-3′, Reverse primer: 5′-CCTCCTGGAAGAACCACCAC-3′. The qRT-PCR primers for GAPDH were as follows: Forward primer: 5′-ACCTGACCTGCCGTCTAGAA-3′, Reverse primer: 5′-TCCACCACCCTGTTGCTGTA-3′.
3
Quantitative RT-PCR for Gene Expression Analysis
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Total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcribed using Reverse Transcription PrimeScript 1st Stand cDNA Synthesis kit (TaKaRa, Otsu, Japan). qRT-PCRs were performed using quantitative PCR reagents SYBR PremixEx TaqTM (TaKaRa) following the manufacturer's instructions. Levels of GAPDH quantified with target genes acts as an internal control and fold-changes were analyzed using the 2−ΔΔCt method. The qRT-PCR primer sequences were shown in Supplementary Table 2 .
4
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcribed using Reverse Transcription PrimeScript 1st Stand cDNA Synthesis kit (TaKaRa, Otsu, Japan). qRT-PCR were performed using quantitative PCR reagents SYBR PremixEx TaqTM (TaKaRa) following the manufacturer's instructions. Levels of GAPDH quantified with target genes acts as an internal control and fold-changes were analyzed using the 2-ΔΔCt method. The qRT-PCR primer sequences were shown in Supplementary Table 2 .
5
SLCO4C1 Methylation and Expression Analysis
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Bisulfite amplicon sequencing (BSAS) was performed within CpG sites of the SLCO4C1 in the same region as the four sites selected from TCGA. The primers specific for SLCO4C1 were forward 5′-TAAGGGGAGTTTATGGTTAGAGAT-3′ and reverse 5′-AAAAARGAAAATTCTCACCCC-3′. Then, MethylKIT software (http://www.bioconductor.org/packages/release/bioc/html/methylKit.html ) was used to analyse methylation data and calculate the average methylation level at all sites. Total RNA was extracted using TRIzol reagent (TaKaRa). cDNA reverse transcription and qRT-PCR were performed following the instructions of Reverse Transcription PrimeScript 1st Stand cDNA Synthesis kit (TaKaRa) and quantitative PCR reagents SYBR Premix ExTaq™ (TaKaRa). β-Actin was used as the corresponding internal control. All mRNA levels were quantified by the 2−ΔΔCT method. Each reaction was performed in triplicate. The primers specific for SLCO4C1 were forward 5′-AGGGGCCATCCACAGTCTTC-3′ and reverse 5′-AAGACCCCTCCTCAAACTCG-3′.
6
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from cells using Trizol reagent (Invitrogen). cDNA reverse transcription, and qRT-PCR were performed as per the manufacturers’ instructions using Reverse Transcription PrimeScript 1st Stand cDNA Synthesis kit (TaKaRa, Otsu, Japan) and quantitative PCR reagents SYBR PremixEx TaqTM (TaKaRa). The relative expression of genes was calculated with the 2−ΔΔCt method. The sequences of the primers used are presented in Table S1 .
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