RNA was extracted from cells using RNEasy (QIAGEN, Hilden, Germany), and reverse transcription was carried out using the iScript cDNA Synthesis Kit (Bio Rad Laboratories) per the manufacturer's instructions. A total of 15 ng cDNA was subjected to qPCR using TaqMan chemistry on the CFX384 Touch Real-Time PCR system (Bio Rad Laboratories). Primers were obtained from Thermo Fisher Scientific (VEGF, #Hs00900055_m1; CHOP, #Hs01090850_m1). Each test was run in triplicate with relative transcript levels of target gene expressed as 2-ΔΔCT with POLR2A (#Hs00172187_m1; Thermo Fisher Scientific) as the housekeeping control gene.
Polr2a
Manufactured by Thermo Fisher Scientific
POLR2A is a gene that encodes the largest subunit of RNA polymerase II, which is responsible for the transcription of protein-coding genes in eukaryotic cells. The product of this gene is a key component of the RNA polymerase II complex, which plays a central role in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Cfx384 touch real time pcr system, by Bio-Rad (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Hprt1, by Thermo Fisher Scientific (1 mentions)
Ifi44, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Ddit3, by Thermo Fisher Scientific (1 mentions)
Hmox1, by Thermo Fisher Scientific (1 mentions)
Taqman master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
5 protocols using polr2a
1
Quantitative Real-Time PCR for Gene Expression
2
Quantifying Immune Gene Expression
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Total RNA was extracted from cells (1–2 × 106 cells per sample) with an RNeasy kit (Qiagen, Germantown, MD, USA) and cDNA was generated using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed on a Light Cycler 480 II (Roche, Indianapolis, IN, USA). Primers for IFIT1 (Hs03027069), IFI44 (Hs00197427), RAGE (Hs00542584), FcRIIa (Hs01013401), MX1 (Hs00182073,), HPRT1 (Hs99999909), and Polr2a (Hs00172187) were purchased from Thermo Scientific. The genes of interest were normalized to the expression of the house keeping gene (Polr2a) and were compared to a control condition with no treatment. The relative induction was calculated by 2−ΔΔCt.
3
Quantitative PCR for c-erb-B2 Expression
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Two-step quantitative reverse transcriptase-mediated real-time PCR (qPCR) was used to measure the relative abundance of c-erb-B2 mRNA from equal amounts of cDNA (10 ng) of B16 and B16/neu using the TaqMan™ gene expression assay (Rn00566561_m1) and POLR2A (Mm00839502_m1) (Thermo Fisher Scientific, Carlsbad, CA). Data were normalized to the endogenous control POLR2A,40 (link) and mRNA abundance was calculated using the ΔΔCT method.40 (link)
4
Quantifying mRNA Expression Profiles
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qRT-PCR for mRNA detection was performed using 10 μl 2x Taqman Universal PCR Master Mix II and 1 μl Taqman gene expression assay (HOXA5 - Cat No. Hs00430330_m1, DUSP3 - Cat No. Hs01115776_m1, RYR1 - Cat No. Hs01062613_m1, p53 - Cat No. Hs01034249_m1 and PTEN - Cat No. Hs02621230_s1, Thermo Fisher Scientific), 8 μl RNase-free water and 1 μl cDNA template (50 ng/μl). Duplicates were run for all reactions together with no template controls and an additional control for DNA contamination where the reverse transcriptase was omitted. As a detection system the ABI Prism 7900 sequence detector (Thermo Fisher Scientific) was programmed to an initial step of 10 min at 95° C, followed by 40 thermal cycles of 15 s at 95° C and 10 min at 60° C. The log-linear phase of amplification was monitored to obtain CT values for each RNA sample. The expression level of the respective mRNAs was analyzed in relation to the levels of housekeeping gene POLR2A (Cat No. Hs00172187_m1, Thermo Fisher Scientific). Conversion of the individual CT values to the linear form was performed according to the 2–∆∆CT method [71 (link)] and the relative standard curve method.
5
Gene Expression Analysis in Cultured Cells
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Total RNA was isolated from cultured cells using RNeasy mini kit, Qiagen (Venlo, Netherlands) and cDNA was produced using Iscript cDNA synethesis kit, Bio-Rad (Hercules, CA; #1708890). qRT-PCR was performed using TaqMan master mix, ThermoFisher (Waltham, MA; #4444557). Primers: DDIT3 (HS00358796_g1), BOK (Hs011006404_m1), NOXA1 (Hs00611456_g1), HMOX1 (#Hs00157965_m1), NQO1 (#Hs01045993_g1), GCLM (#Hs00978072_m1), NFE2L2 (Hs00975961_g1), and POLR2A (Hs00172187_m1) were purchased from ThermoFisher. POLR2A was used as the endogenous control. The relative fold change was calculated based on the formula R = 2−(ΔCt sample − ΔCt control).
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