Following homogenization of the cell lysates using Qiashredder columns (Qiagen) and removal of genomic DNA with a gDNA column (Qiagen), RNA was isolated from drug- and control-treated cells using an RNeasy mini kit (Qiagen) and reverse transcribed using the first strand cDNA synthesis system (Invitrogen). Quantitative PCR was performed with SYBR mix (BioRad, Hercules, CA) using PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA-specific primers (Supplementary Table S1 ) and POLR2A-specific primers (Supplementary Table S1 ) as an internal standard using an iCycler (BioRad) according to the manufacturer’s instructions. All reactions were performed in triplicate, and the experiment was repeated three times. Relative expression levels and standard deviations were calculated using the comparative method (Applied Biosystems, Grand Island, NY).
Gdna column
Manufactured by Qiagen
The GDNA column is a laboratory equipment designed for the purification of genomic DNA (gDNA) from various sample types. It utilizes a silica-based membrane technology to efficiently capture and concentrate gDNA, allowing for its subsequent elution in a clean and ready-to-use format.
Automatically generated - may contain errors
Lab products found in correlation
First strand cdna synthesis system, by Thermo Fisher Scientific (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Icycler, by Bio-Rad (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Rneasy miniprep kit, by Qiagen (1 mentions)
2 protocols using gdna column
1
Quantitative PCR Analysis of Apoptosis Genes
2
RNA Isolation, cDNA Synthesis, and qPCR Analysis
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For RNA isolations, cells were lysed in RLT buffer containing 1% b-mercaptoethanol.
Genomic DNA was removed via gDNA column (Qiagen, Valencia, CA), and RNA was isolated with an RNeasy mini prep kit (Qiagen). Complementary DNA was generated using high capacity cDNA kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using Taqman (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.24.380527 doi: bioRxiv preprint Master Mix and Taqman probes as listed in Table 2. RNA content was normalized to GAPDH and expression levels were quantified using 2 -ΔΔCT method from at least three independent experiments performed in triplicate.
Genomic DNA was removed via gDNA column (Qiagen, Valencia, CA), and RNA was isolated with an RNeasy mini prep kit (Qiagen). Complementary DNA was generated using high capacity cDNA kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using Taqman (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.24.380527 doi: bioRxiv preprint Master Mix and Taqman probes as listed in Table 2. RNA content was normalized to GAPDH and expression levels were quantified using 2 -ΔΔCT method from at least three independent experiments performed in triplicate.
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