Total RNA was extracted from the ESCC tissues and ESCC cell lines using the miRNA or mRNA Extraction Kit (Qiagen, Hilden, Germany) according to the protocol of the manufacturer. The cDNA of miRNA or mRNA were synthesized with One Step PrimeScript miRNA or mRNA cDNA Synthesis Kit (Qiagen). Quantitative real-time PCR (qPCR) was performed using the SYBR green Premix Ex Taq II (Qiagen) with StepOne Plus Real-Time PCR System (Applied Biosystems). The expression of U6 was used as endogenous control for detection of miRNA expression level. The expression of β-actin was used as endogenous control for detection of mRNA expression level. The reaction was performed on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The 2−ΔΔCt method was used to quantify the expression of miR-145 and PLCE1.
Sybr green premix ex taq 2
Manufactured by Qiagen
SYBR green Premix Ex Taq II is a real-time PCR master mix that contains SYBR Green I dye, optimized buffer, and Taq DNA polymerase. It is designed for sensitive and specific detection of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Primer script reverse, by Takara Bio (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mirna extraction kit, by Qiagen (1 mentions)
Mirna cdna synthesis kit, by Qiagen (1 mentions)
3 protocols using sybr green premix ex taq 2
1
Quantifying miR-145 and PLCE1 Expression
2
Quantitative PCR analysis of Nephrin gene
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Total RNA from CIHP-1 cells seeded into a 6-well plate (6x104 cells/well) was extracted using TRIzol® reagent (Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the PrimerScript reverse transcriptase (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. qPCR analysis was performed on a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR green Premix Ex Taq II (Qiagen GmbH), according to the manufacturer's protocol. The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 5 min; followed by 40 cycles of denaturation at 95 ˚C for 45 sec, annealing at 50˚C for 45 sec and elongation at 72˚C for 45 sec; and a final extension step at 72˚C for 10 min. GAPDH was used as an internal reference gene and the relative gene expression was determined using the 2-ΔΔCq method (24 (link)). The following primer pairs were used for qPCR: Nephrin forward, 5'-CTGCCTGAAAACCTGACGGT-3' and reverse, 5'-GACCTGGCACTCATACTCCG-3'; and GAPDH forward, 5'-TGTGGGCATCAATGGATTTGG-3' and reverse, 5'-ACACCATGTATTCCGGGTCAAT-3'.
3
Quantitative Analysis of miR-34a in ESCC
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Total RNA was isolated from 78 cases of ESCC tissues, 25 cases of non-tumor tissues, and ESCC cell lines transfected with miR-34a mimic, inhibitor, si-PLCE1, and their own blank vector using a miRNA Extraction Kit (Qiagen, Hilden, Germany). Among the tissue samples, there were 25 paired ESCC tissues and their corresponding non-tumor tissues were selected randomly. Reverse transcription for the quantification of miRNAs was conducted via miRNA cDNA Synthesis Kit (Qiagen). The qPCR amplification of miR-34a was executed via SYBR green Premix Ex Taq II (Qiagen) using Step One Plus Real-Time PCR System (Applied Biosystems). The expression level of miRNA was normalized using U6 as an internal control. The experiment was executed in ABI Prism 7500 Sequence Detection System. The reaction was carried out with an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The expression level of miR-34a gene in all cases was calculated by 2−ΔCt method, ΔCt = Ct miR-34a - Ct U6. The difference of miR-34a expression in ESCC and corresponding normal tissues was represented by the RQ value, which was 2−ΔΔCt (ΔΔCt = ΔCt ESCC-ΔCt corresponding normal tissues), indicating the significant miR-34a gene expression of ESCC in tissues compared with adjacent normal tissue. Primers were used as follows:
MiR-34a: 5′-CCCAGAACATAGACACGCTGGA-3′
U6: 5′-TGGTGAAGACGCCAGTGGA-3′
MiR-34a: 5′-CCCAGAACATAGACACGCTGGA-3′
U6: 5′-TGGTGAAGACGCCAGTGGA-3′
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