Total RNA was extracted from the cells (or tissues) using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For miR-125a-5p quantitative PCR, cDNA was synthesized with TaqMan MicroRNA hsa-miR-125a-5p specific primers (Applied Biosystems) using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems). qRT-PCR was performed in duplicate using QuantiTect SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and analyzed on an ABI Prism 7500 analyzer (Applied Biosystems, Foster City, CA). The primers used were as follows: A20, forward, 5′-AAAGCCCTCATCGACAGAAA -3′ , reverse, 5′-CAGTTGCCAGCGGAATTTA-3′ ; TRAIL, forward, 5′-accaacgagctgaagcagat-3′ , reverse, 5′-cagcaggggctgttcatact-3′ ; and GAPDH, forward, 5′-GGAGTCAACGGATTTGGT-3′ , reverse, 5′-GTGATGGGATTTCCATTGAT-3′ .
Abi prism 7500 analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7500 analyzer is a real-time PCR system designed for quantitative gene expression analysis and genotyping. It features a 96-well thermal cycler with a configurable optical system for monitoring fluorescence signals during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Reverse transcriptase kit, by Thermo Fisher Scientific (2 mentions)
Cm0451, by Procell (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Qiagen (1 mentions)
Sybr green pcr mixture, by Qiagen (1 mentions)
4 protocols using abi prism 7500 analyzer
1
Quantification of miR-125a-5p and target genes
2
siRNA-Mediated Knockdown of ESM1 in Human ACC
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The siRNA treatment was conducted as previously described (42 (link)). The human SW13 cell, one of the human ACC cell lines, was transfected with 50 nmol of ESM1 siRNA (siESM1) or negative control siRNA (siNC) in a special medium (CM0451, Procell, Wuhan, China) for 48 h. SW13 cells were then lysed by TRIzol reagent (Invitrogen, Waltham, MA, USA) for total RNA isolation. The cDNA was obtained by oligo-dT primers and reverse transcriptase kit (Invitrogen, USA). Quantitative real-time PCR (qRT-PCR) was performed by SYBR Green PCR Master Mix (Qiagen, Hilden, Germany) and specific primers in an ABI Prism 7500 analyzer (Applied Biosystems, Waltham, MA, USA). GAPDH was an endogenous reference gene. Three replicates were set for all reactions. The 2−ΔΔCt method was applied to calculate the relative expression of ESM1 in ACC samples. The related primers are listed in Supplementary Table S4
3
siRNA Knockdown of CENPF and CDK1 in ACC
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The siRNA transfections were performed according to the manufacturer’s instructions. As one of the human ACC cell lines, SW13 cells were transfected with 50 nmol of CENPF siRNA (siCENPF), CDK1 siRNA (siCDK1), or negative control siRNA (siNC) in a special medium (CM0451, Procell, China) for 48 h. Then, SW13 cells were lysed by TRIzol reagent (Invitrogen, USA) for total RNA isolation. The cDNA was obtained by reverse transcriptase kit (Invitrogen, USA). SYBR Green PCR Mixture (Qiagen, Germany) and specific primers were performed in ABI Prism 7500 analyzer (Applied Biosystems, USA). GAPDH was an endogenous reference gene. Three replicates were set for all reactions. The 2−ΔΔCt method was applied to calculate the relative expression of CENPF or CDK1 in ACC samples. The details of primers were listed in Additional file 4 : Table S4.
4
Quantitative Real-Time PCR Protocol for SCLC
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Quantitative real-time PCR (RT-qPCR) were performed according to methods published previously [23 (link), 24 (link)]. Total RNA was extracted from the PPFE samples by using RNeasy FFPE Kit (cat. no. 73504, QIAGEN, Germany). Total RNA of 1 μg was reversely transcribed in a 20 μl reaction using RevertAid First Strand cDNA Synthesis Kit (cat. no. #K1622, Thermo Scientific, USA) according to the manufacturer’s protocol. The reaction products were then diluted with 40 μl RNase-free water. The real-time PCR reaction was composed of 2 μl cDNA, 10 μl of PowerUp™ SYBR™ Green Master Mix (cat. no. A25741, Thermo Scientific, USA) and 0.5 μl of forward and reverse primers (0.5 μM). RT-qPCR was conducted in an ABI Prism 7500 analyzer (Applied Biosystems, USA) for 40 cycles (95°C for 15 sec, 58°C for 15 sec, 72°C for 30 sec) after an initial 120s denaturation at 95°C. HPRT1 was endogenous reference gene. All reactions were run in triplicate. The relative RNA levels of SCLC samples were calculated by using the 2−ΔΔCt method. All primers of the hub genes and HPRT1 were synthesized by Sangon Biotech (Shanghai, China), and the information of their sequences were listed in Table 2 .
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