List of differentially expressed genes in human endometrial stromal cells with knockdown of Basigin (BSG) gene expression during decidualization.
The BSG siRNA or negative scrambled control siRNA were transfected into human endometrial stromal cells (HESCs) following the protocol of siLentFect™ Lipid (Bio-Rad, Hercules, CA. Following complete knock down of BSG in HESCs (72 hours after adding siRNA), HESCs were treated with medium containing estrogen, progesterone and cAMP to induce decidualization. BSG siRNA and negative control scrambled siRNA were added to the cells every four days (day 0, 4) over the course of the decidualization protocol. Total RNA was harvested at day 6 of the decidualization protocol for microarray analysis. Microarray analysis was performed at the University of Illinois at Urbana-Champaign Roy J. Carver Biotechnology Center. Briefly, 0.2 micrograms of total RNA were labeled using the Agilent two color QuickAmp labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s protocol. The optional spike-in controls were not used. Samples were hybridized to Human Gene Expression 4x44K v2 Microarray (Agilent Technologies, Santa Clara, CA) in an Agilent Hybridization Cassette according to standard protocols. The arrays were then scanned on an Axon GenePix 4000B scanner and the images were quantified using Axon GenePix 6.1.
Microarray data pre-processing and statistical analyses were done in R (v3.6.2) using the limma package (3.42.0 (Ritchie et al., 2015). Median foreground and median background values from the 4 arrays were read into R and any spots that had been manually flagged (-100 values) were given a weight of zero. The background values were ignored because investigations showed that trying to use them to adjust for background fluorescence added more noise to the data; background was low and even for all arrays, therefore no background correction was done.
The individual Cy5 and Cy3 fluorescence for each array were normalized together using the quantile method 3 (Yang and Thorne, 2003). Agilent's Human Gene Expression 4x44K v2 Microarray has a total of 45,220 probes: 1224 probes for positive controls, 153 negative control, 823 labeled “ignore” and 43,118 labeled “cDNA”. The pos+neg+ignore probes were used to ascertain the background level of fluorescence (6, on the log2 scale) then discarded. The cDNA probes comprise 34,127 unique 60mer probes, of which 999 probes are spotted 10 times each and the rest one time each. We averaged the replicate probes for those spotted 10 times and then fit a mixed model that had treatment and dye as fixed effects and array pairing as a random effect (Phipson et al., 2016; Smyth et al., 2005). After fitting the model but before False Discovery Rate (FDR) correction (Benjamini and Hochberg, 1995), probes were filtered out by the following criteria: 1) did not have at least 4/8 samples with expression values > 6 (14,105 probes removed), 2) no longer had an assigned Entrez Gene ID in Bioconductor’s HsAgilentDesign026652.db annotation package (v3.2.3; 2,152 probes removed) (Huber et al., 2015), 3) mapped to the same Entrez Gene ID as another probe but had a larger p-value for treatment effect (4,141 probes removed). This left 13,729 probes representing 13,729 unique genes.
<b>*Please note: that there is a discrepancy between the file and the readme as this plain text is the actual data file of this dataset.</b>