Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells

Abstract

The imbalance in homocysteine (Hcy) metabolism has been implicated in the pathogenesis of human diseases, including cardiovascular and neurodegenerative disorders. When attempting to identify gene expression profiles using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), the selection of suitable reference genes is important. Here, the expression levels of 10 commonly used reference genes were assessed for normalization of RT-qPCR in Hcy-treated human umbilical vein endothelial cells (HUVECs) and control cells. The suitability of eight selected candidate genes was comparatively analyzed across the tested samples and separately ranked by four programs, geNorm, NormFinder, BestKeeper, and the ΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the most stable gene in the final ranking using the RankAggreg package. Surprisingly, the β-actin (ACTB) levels decreased significantly in Hcy-treated HUVECs compared with control HUVECs (P<0.05), and further study indicated that Hcy suppressed the expression of ACTB by upregulating the miR-145-5p level in Hcy-treated HUVECs. Our data suggest that GAPDH can be used as a reliable reference gene, while ACTB cannot; normalization of gene expression in RT-qPCR experiments in Hcy-treated HUVECs. The data, which identifies a suitable reference gene in Hcy-treated HUVECs, will contribute to the design of an effective and accurate method for quantitation of gene expression.

The crucial choice of reference genes: identification of miR-191-5p for normalization of miRNAs expression in bone marrow mesenchymal stromal cell and HS27a/HS5 cell lines

“…As suggested by Andersen et al 66 , a minimum of 5 candidates reference genes should be analysed. miR-16-5p, miR-103a-3p and miR-191-5p were chosen because of their reported expression in BM-MSCs 23 or their use in stability analysis in other models [67][68][69][70] . miR-21-5p was chosen for validation experiment since it is described to be expressed in BM-MSCs and could be used as BM-MSC marker 23 .…”

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

“…For example, statin-treated HUVEC cells showed that HPRT1 and YWHAZ were considered the most suitable reference genes 29 . However, in homocysteine-treated HUVEC cells, YWHAZ was not stably expressed and ranked seven of eight 21 . In addition, hydrogen peroxide-treated HUVEC showed HPRT1 was a less reliable reference gene with a ranking of 11 of 15 20 .…”

“…Multiple studies have investigated candidate reference genes in iPSCs or ECs 10,19−23 , during reprogramming and differentiation 10,23 . In addition, various publications reported a selection of housekeeping genes in endothelial cells, such as microvascular EC 19 , human retinal EC 21 , and human blood-brain barrier EC 22 . The candidate reference genes examined in these studies were among the commonly used housekeeping genes in our study, including B2M, GAPDH, GUSB, HPRT1, HMBS, and YWHAZ.…”

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

“…Multiple studies have investigated candidate reference genes in iPSCs or ECs 18 , 29 – 33 , during reprogramming and differentiation 18 , 33 . In addition, various publications reported a selection of housekeeping genes in endothelial cells, such as microvascular EC 29 , human retinal EC 31 , and human blood–brain barrier EC 32 . The candidate reference genes examined in these studies were among the commonly used housekeeping genes in our study, including B2M , GAPDH , GUSB , HPRT1 , HMBS , and YWHAZ .…”