Validation of Housekeeping Genes as Reference for Reverse-Transcription-qPCR Analysis in Busulfan-Injured Microvascular Endothelial Cells

Abstract

Endothelial cells (ECs) could express some important cytokines and signal molecules which play a key role in normal hematopoiesis and repopulation. Busulfan-induced vascular endothelial injury is an important feature after hematopoietic stem cell transplantation (HSCT). But the molecular mechanism of how the injured ECs affect hematopoietic reconstruction is still unknown. It is possibly through modulation of the change of some gene expression. RT-qPCR is one of the most popular methods used to accurately determine gene expression levels, based on stable reference gene (RG) selection from housekeeping genes. So our aim is to select stable RGs for more accurate measures of mRNA levels during Busulfan-induced vascular endothelial injury. In this study, 14 RGs were selected to investigate their expression stability in ECs during 72 hours of EC injury treated with Busulfan. Our results revealed extreme variation in RG stability compared by five statistical algorithms. ywhaz and alas1 were recognized as the two idlest RGs on account of the final ranking, while the two most usually used RGs (gapdh and actb) were not the most stable RGs. Next, these data were verified by testing signalling pathway genes ctnnb1, robo4, and notch1 based on the above four genes ywha, alas1, gapdh, and actb. It shows that the normalization of mRNA expression data using unstable RGs greatly affects gene fold change, which means the reliability of the biological conclusions is questionable. Based on the best RGs used, we also found that robo4 is significantly overexpressed in Busulfan-impaired ECs. In conclusion, our data reaffirms the importance of RGs selection for the valid analysis of gene expression in Busulfan-impaired ECs. And it also provides very useful guidance and basis for more accurate differential expression gene screening and future expanding biomolecule study of different drugs such as cyclophosphamide and fludarabine-injured ECs.

A novel strategy for isolation of mice bone marrow endothelial cells (BMECs)

“…Primer sequences and probes are shown in (Table 2). For RT-qPCR, the synthesized cDNA samples 10 ng were amplified with the SYBR green master mix in a final volume of 20 ul, as described in our previously published article [18]. The mean threshold values are used to evaluate the molecular gene expression with normalization with mouse betaactin and GAPDH.…”

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 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.…”

E4orf1 Suppresses E1B-Deleted Adenovirus Vaccine-Induced Immune Responses

“…No PCR product appearing on the total RNA gel showed that no genomic DNA contaminated the total RNA. Product integrity was also evaluated using 0.8% agarose gel electrophoresis and showed no contamination with DNA or degradation of RNA [23,24].…”