TY - JOUR
T1 - Selection of reference genes for studies of human retinal endothelial cell gene expression by reverse transcription-quantitative real-time polymerase chain reaction
AU - Appukuttan, Binoy
AU - Ashander, Liam
AU - Ma, Yuefang
AU - Smith, Justine
PY - 2018/3
Y1 - 2018/3
N2 - Background Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. Methods Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) < 0.2 and coefficient of variation (CV-value) < 0.1. Results Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11–21 (41–78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. Conclusions We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells.
AB - Background Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. Methods Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) < 0.2 and coefficient of variation (CV-value) < 0.1. Results Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11–21 (41–78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. Conclusions We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells.
KW - Endothelial cell
KW - Human
KW - PCR
KW - Reference gene
KW - Retina
UR - http://www.scopus.com/inward/record.url?scp=85037630612&partnerID=8YFLogxK
U2 - 10.1016/j.genrep.2017.11.009
DO - 10.1016/j.genrep.2017.11.009
M3 - Article
VL - 10
SP - 123
EP - 134
JO - Gene Reports
JF - Gene Reports
SN - 2452-0144
ER -