We extracted all RNAs from kidney tissues and HK-2 cells using TRIzol reagent (Takara, Shiga, Japan) and a total RNA isolation kit (Takara, Japan). RNA samples were reverse-transcribed into complementary DNA using an RT Primer (Takara, Japan). The relative levels of target gene to the control GAPDH mRNA transcripts were quantified in triplicate by RT-qPCR using specific primers. The generated data were analyzed using the 2-△△Ct method.
Rt primer
Manufactured by Takara Bio
Sourced in Japan
RT primer is a short, single-stranded DNA molecule used in reverse transcription reactions. It serves as a starting point for the synthesis of complementary DNA (cDNA) from RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Total rna isolation kit, by Takara Bio (1 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Reverse transcriptase, by Takara Bio (1 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Recombinant rnase inhibitor, by Takara Bio (1 mentions)
Poly a polymerase, by Takara Bio (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dmem high glucose medium, by Procell (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Forward primer, by Takara Bio (1 mentions)
Dual luciferase reporter gene kit, by Promega (1 mentions)
Dmem f12 medium, by Procell (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Abi prism 7900, by Thermo Fisher Scientific (1 mentions)
Sybr pcr master mix, by Takara Bio (1 mentions)
6 protocols using rt primer
1
Quantitative Gene Expression Analysis
2
Cardiac Tissue miRNA and mRNA Expression
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Total RNA was extracted from cardiac tissue or H9c2 cells using TRIzol® reagent (Invitrogen; USA). Reverse transcription of RNA was performed with PrimeScript RT kit (TAKARA, Japan) according to the kit instructions. Reverse transcription of miRNA was carried out with Poly A polymerase (TAKARA, Japan), RT primer, recombinant RNase inhibitor (TAKARA, Japan), reverse transcriptase (TAKARA, Japan), rATP and dNTPs according to the manufacturer’s protocol. PCR reaction systems were prepared using SYBR® Premix Ex TaqTM (TAKARA, Japan) according to the kit instructions. The primers are listed in Table 1 . MiRNA and mRNA expression levels were quantified by the 2-ΔΔCt method. GAPDH was used as an internal control for mRNAs and U6 was used for miRNA.
3
miRNA Expression Profiling Protocol
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The following materials were used in the study: DMEM/F12 medium (Procell, pm150210), DMEM high glucose medium (Procell, 150,310), fetal bovine serum (Gibco, 10,099,141), trypsin, streptomycin, and penicillin (Gibco). The miRNA qRT-PCR Starter Kit (TakaRa), forward primer (TakaRa), RT primer (TakaRa), and miRNA mimics (genephma) were used. Lipofectamine™ 3000 (Invitrogen) was employed for transfection. The dual luciferase reporter gene kit and reporter gene vector were obtained from Promega.
4
Quantifying Gene Expression by qPCR
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Total RNA was isolated from tissue samples and cell lines via TRIZOL reagent (Invitrogen, USA) and was synthesized into complementary (cDNA) via the Reverse Transcription Kit with specific RT-primers (Takara, Japan). Quantitative PCR was performed on the ABI Prism 7900 (Applied Biosystems, USA) using the SYBR PCR master mix (Takara, Japan) with specific PCR primers, listed in Table S1 . The relative expression of individual genes was analyzed by the 2-△△Ct method46 (link). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control for KCNQ1TO1 and mRNA expression, and U6 for miRNAs expression.
5
miRNA Expression Analysis in MSCs
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Perform total RNA extraction and qRT-PCR analysis according to the manufacturer’s protocol. We used TRIzol reagent (Synthgene, Nanjing, China) to extract total RNA from MSCs at the designated time points. We used Taqman probes (Applied Biosystems, USA) to quantify miRNA. In short, RT primers and AMV reverse transcriptase (Takara, Japan) were used to transcribe total RNA into cDNA. Subsequently, real-time PCR was performed on the Applied Biosystems 7300 sequence detection system using the Taqman PCR kit (Applied Biosystems, USA). The reaction conditions were: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and then 60°C for 1 min. The 2-ΔΔCT method was used for quantitative determination, and the expression of GAPDH was used as an internal control.
6
RNA Extraction and Quantitative PCR
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Total RNA was extracted by RNAiso Plus (Takara). We used 1 μg of total RNA per reaction for the first-strand cDNA synthesis using RT primers (Takara). Quantitative real-time PCR assays were performed using cDNA in a 10 μL reaction volume (SYBR Green PCR kit; Takara) on an Applied LightCycler 480 system (Roche). Gene expression levels were measured using the ΔΔCt method with normalization to β-actin. The primers used in this study were obtained from Sangon (primer sequences were listed in Tables 1 , 2 ).
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