qRT-PCR was used to detect the RNA expression of METTL14 and miR-1306-5p. All the RNA was collected from blood, cell, or tissue samples using the miRNeasy extraction kit (QIAGEN). For quantitative analysis, the cDNA was reversed by miRNA Reverse Transcription Kit (MR101-01/02, Vazyme) and detected by all-in-one miRNA RT-qPCR Detection Kit (Q711-02, Vazyme) with U6 as the internal control. As for METTL14 mRNA detection, TRIzol (BS259A, Biosharp) was used for RNA isolation and PrimeScript RT Reagent kit (R223-01, Vazyme) was used to reverse RNA into cDNA. SYBR Green Real-Time PCR Master Mix (Q711-02, Vazyme) was used for RT-PCR assay with β-actin as the control. The primers for miR-1306-5p, U6, METTL14, and β-actin were listed as below: METTL14, sense, 5′- GAGTGTGTTTACGAAAATGGGGT-3′; antisense, 5′- CCGTCTGTGCTACGCTTCA-3′; β-actin: sense, 5′-AGCGAGCATCCCCCAAAGTT-3′, antisense: 5′-GGGCACGAAGGCTCATCATT-3′; U6: sense, 5′-CTCGCTTCGGCAGCACA-3′, antisense: 5′-AACGCTTCACGAATTTGCGT-3′; miR-1306-5p reverse primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTGGACGTT-3′; miR-1306-5p sense: 5′-AATACCACCTCCCCTGCA-3′. 2−ΔΔCt method was used for analysis of relative expression.
Mirna reverse transcription kit
Manufactured by Vazyme
Sourced in China
The MiRNA reverse transcription kit is a laboratory tool designed to perform reverse transcription of miRNA (microRNA) molecules. It converts miRNA into complementary DNA (cDNA) for further analysis and downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Sybr green real time pcr master mix, by Vazyme (1 mentions)
Primescript rt reagent kit, by Vazyme (1 mentions)
Mirneasy extraction kit, by Qiagen (1 mentions)
Trizol, by Biosharp (1 mentions)
Mrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green pcr kit, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Sybr green supermix, by Bio-Rad (1 mentions)
5 protocols using mirna reverse transcription kit
1
Quantifying METTL14 and miR-1306-5p in Biological Samples
2
Liver miRNA Extraction and Quantification
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Total RNA from the liver samples was extracted using a Total RNA kit (Forgene, Sichuan, China) according to the manufacturer’s instructions. A miRNA reverse transcription kit (Vazyme, Jiangsu, China) and a first-strand cDNA synthesis kit (Forgene, Sichuan, China) were applied to synthesize cDNA. Reverse transcriptase quantity PCR (RT-qPCR) was accomplished using SYBR Green PCR Master Mix (Forgene, Sichuan, China). Primers for RT-qPCR are presented in Supplementary Table S1 . The same reverse primer with the sequence 5′-AGTGCAGGGTCCGAGGTATT-3′ was used for all miRNAs. The average expression levels of liver miRNAs and mRNAs were normalized to U6 and GAPDH, respectively. Relative gene expression was determined by the 2−ΔΔCt method.
3
RNA Extraction and Expression Analysis
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The RNAeasy™ animal RNA extraction kit (cat. no. R0026, Beyotime Institute of Biotechnology) was used to extract total intracellular RNA. mRNA and miRNA were reverse transcribed into cDNA using a mRNA reverse transcription kit (Thermo Fisher Scientific, Inc.) and miRNA reverse transcription kit (Vazyme Biotech Co., Ltd.). SYBR-Green PCR Master Mix (Thermo Fisher Scientific, Inc.) was used for RT-qPCR. Levels of miRNA were normalized using small nuclear RNA U6, whereas ADAM12 levels were normalized by GAPDH. The 2-ΔΔCq method was used to calculate the difference in gene expression (27 (link)). The primers were synthesized by Tsingke Co., Ltd. The specific sequences of the primers are shown in Table SIII .
4
Quantification of miRNA and mRNA Expression
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Trizol reagent (Invitrogen) was used for total RNA isolation. Quantification of miR-582-5p was performed as described previously [14 (link)]. cDNA was reverse-transcribed from RNA using the miRNA Reverse Transcription kit (Vazyme Biotech, Nanjing, China). The primers were as follows: miR-582-5p, forward: 5′-GCACACATTGAAGAGGACAGAC-3′, reverse: 5′-TATTGAAGGGGGTTCTGGTG-3′. For analysis of mRNA expression, cDNA was synthesized using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). PCR was performed using the iTaq Universal SYBR Green PCR Kit (BioRad). The primers were as follows: CMTM8, forward: 5′-GGAGGAGCCGCAGCGCG-3′, reverse: 5′-CTGTATGGTCCTGGATCTCC-3′; KLF15, forward: 5′-ATGCACAAATGTACTTTCCCT-3′, reverse: 5′-TCAGTTCACGGAGCGCACGGA-3′; FOXG1, forward: 5′-CTTCATCCTGAGTCCCTACCG-3′, reverse: 5′-GCCGTTCTGCTGCATTCG-3′. Relative gene expression was analyzed using the 2−ΔΔCT method [19 (link)].
5
miRNA Reverse Transcription and qPCR
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For cDNA synthesis of miRNA, reverse transcription was carried out using the miRNA reverse transcription kit (Vazyme) with the miR17/miR16/let-7a/U6 stem-loop primer (Supplementary Table S12 ). The primer was designed using miRNA Design V1.01 software. Briefly, 1 μg total RNA collected from 293T cells was mixed with 100 nM stem–loop primer and the RT SuperMix (containing dNTP, reaction buffer, reverse transcriptase and RNase inhibitor). The reaction was conducted at 25°C for 5 min and the 50°C for 15 min, followed by the inactivation of the reverse transcriptase at 85°C for 5 min. The qPCR experiments were carried out in the reaction mixture containing SYBR Green Supermix (Bio-Rad), 0.2 μM forward primer, 0.2 μM reverse primer and diluted cDNA using the CFX96 Real-Time system (Bio-Rad). The thermocycling condition was: 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The U6 small nuclear RNA was used as the reference gene to normalize the miR17/miR16/let-7a expression level. The purity of the PCR product was validated by the electrophoresis gel and the melting profile. The qPCR results were analysed as described above. The sequence information of the primers was listed in Supplementary Table S13 .
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