Identification of stable housekeeping genes for induced pluripotent stem cells and -derived endothelial cells for drug testing

Abstract

There are no validated housekeeping genes in induced pluripotent stem cells (iPSC) and derived endothelial iPSC (iPSC-EC). Thus a comparison of gene expression levels is less reliable, especially during drug treatments. Here, we utilized transcriptome sequencing data of iPSC and iPSC-EC with or without CRISPR-Cas9 induced translocation to identify a panel of 15 candidate housekeeping genes. For comparison, five commonly used housekeeping genes (B2M, GAPDH, GUSB, HMBS, and HPRT1) were included in the study. The panel of 20 candidate genes were investigated for their stability as reference genes. This panel was analyzed and ranked based on stability using five algorithms, delta-Ct, bestkeeper, geNorm, Normfinder, and Reffinder. Based on the comprehensive ranking of Reffinder, the stability of the top two genes—RPL36AL and TMBIM6, and the bottom two genes—UBA1 and B2M, were further studied in iPSC-EC with and without genetic manipulation, and after treatment with telatinib. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), it was shown that gene expression of the top two housekeeping genes, RPL36AL and TMBIM6, remained stable during drug treatment. We identified a panel of housekeeping genes that could be utilized in various conditions using iPSC and iPSC-derived endothelial cells as well as genetically modified iPSC for drug treatment.

Contact lenses as novel tear fluid sampling vehicles for total RNA isolation, precipitation, and amplification

“…The use of housekeeping genes, as a normalization factor in gene expression studies is considered the most accurate method for correcting potential biases in sample collection, starting material, reverse transcriptase, RT-PCR efficiency, and nucleic acid preparation quality [60][61][62] . However, no single gene can serve as a universal endogenous control for all experimental conditions [60][61][62] .…”

“…The use of housekeeping genes, as a normalization factor in gene expression studies is considered the most accurate method for correcting potential biases in sample collection, starting material, reverse transcriptase, RT-PCR efficiency, and nucleic acid preparation quality [60][61][62] . However, no single gene can serve as a universal endogenous control for all experimental conditions [60][61][62] . Of the housekeeping genes we investigated using our RNA isolation method with RT-PCR, GAPDH yielded the highest Ct value, while ACTB had a moderately acceptable Ct value, and HPRT1 did not meet the detection threshold.…”

“…GAPDH's gene network may have a greater potential for specificity than ACTB and HPRT1 towards the discovery of novel interaction changes and treatment targets for ocular disorders. Gene interaction refers to the combined role of several genes in determining phenotypic variability that affects biological processes [60][61][62][63][64] . This could be notable for infections and other anterior segment diseases, as we have reported through our use of the PCViz and Open Targets Platform.…”

“…At least two to three genes are required to validate and test candidate endogenous controls to ensure their suitability for the specific experiments being conducted. Based on previous gene expression reports and potential for human clinical translation 26 , 60 – 62 , 77 , 78 , we chose the following three TaqMan (ThermoFisher) preamplification primers for rabbit species in the present study: glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Oc03823402_g1), actin beta (ACTB, Oc03824857_g1), and hypoxanthine phosphoribosyltransferase 1 (HPRT1, Oc03399461_m1). We then used the PCViz and Open Targets Platform to analyze the translational scope of these genes in ocular tear fluid research.…”