Senescence focused gene arrays in MSC under different oxygen pressures
Description
Expression of senescence associated genes (N=84) was screened in cultures of mesenchymal stromal cells from human healthy donors (N=4) simultaneously isolated and expanded in different oxygen pressure (5% and19.95%). We hypothesized that higher oxygen pressure would increase the expression of genes involved in cellular senescence. RNA from passage 3 cultures at 70% confluence during growth log phase RNA (Qiagen RNeasy) was transcribed using Qiagen RT2 First Strand kit, followed by amplification using Qiagen RT2 Profiler PCR Array focused on cellular senescence and Qiagen SBR Green PCR master mix. Data analysis was performed using the RT2 PCR Profiler PCR Array Data Analysis software provided by Qiagen. Data analysis was based on the ΔΔCT method with normalization of the raw data to housekeeping genes.We found only four genes that were differentially expressed (CDK4, MYC, P53BP1 and SOD2) with 2 fold change (p value less than 0.05). The array did not identify a specific senescence pathway. The samples were from primary cultures and in a small number, therefore data require validation using larger number of donors.
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Steps to reproduce
RNA from passage 3 cultures at 70% confluence during growth log phase RNA (Qiagen RNeasy) was transcribed using Qiagen RT2 First Strand kit, followed by amplification using Qiagen RT2 Profiler PCR Array focused on cellular senescence and Qiagen SBR Green PCR master mix. We used QuantStudio 4 Realtime PCR System (Applied Biosystems). PCR data analysis was performed using the RT2 PCR Profiler PCR Array Data Analysis software provided by Qiagen. Data analysis was based on the ΔΔCT method with normalization of the raw data to housekeeping genes.