Selection of reference genes for use in quantitative reverse transcription PCR assays when using interferons in U87MG

Abstract

Relative gene quantification by quantitative reverse transcription PCR (qRT-PCR) is an accurate technique only when a correct normalization strategy is carried out. Some of the most commonly genes used as reference have demonstrated variation after interferon (IFN) treatments. In this work we evaluated the suitability of seven reference genes (RGs) [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), β-2Microglobulin (B2M), ribosomal RNA subunits 18S and 28S, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and the RNA helicase (DDX5)] for use in qRT-PCR assays in the glioblastoma-derived cell line U87MG treated with IFNα, IFNγ or a co-formulated combination of both IFNs (HeberPAG); untreated cell lines were included as control. Data was analyzed using geNorm and NormFinder softwares. The expression stability of the seven RGs decreased in order of DDX5/GAPDH/HMBS, 18S rRNA, YWHAZ, 28S rRNA and B2M. qRT-PCR analyses demonstrated that DDX5, GAPDH and HMBS were among the best stably expressed markers under all conditions. Both, geNorm and NormFinder, analyses proposed same RGs as the least variables. Evaluation of the expression levels of two target genes utilizing different endogenous controls, using REST-MCS software, revealed that the normalization method applied might introduce errors in the estimation of relative quantities. We concluded that when qRT-PCR is designed for studies of gene expression in U87MG cell lines treated with IFNs type I and II or their combinations, the use of all three GAPDH, HMBS and DDX5 (or their combinations in pairs) as RGs for data normalizations is recommended.

Antipsychotic Drugs Differentially Affect mRNA Expression of Genes Encoding the Neuregulin 1-Downstream ErbB4-PI3K Pathway

“…For each sample, the C q value (quantification cycle) was calculated with the help of Mx-Pro QPCR Software (Agilent Technologies). The C q values of the cDNA sample for a tested gene were averaged and normalized against the reference gene, GAPDH (encoding glyceraldehyde 3-phosphate dehydrogenase) [14] . The data were analyzed by comparative C q method [15] , and the results were expressed as relative gene expression calculated as 2 -ΔΔCq .…”

Selection of internal references for qRT-PCR assays of human hepatocellular carcinoma cell lines

“…Previous studies have always used an arbitrary method of preselection of reference genes to be evaluated [ 2 , 12 , 14 , 19 , 47 ], thereby leading to missing of potential reference genes. Because of the availability of high-throughput transcriptome profiling technologies, such as gene expression microarray and RNA-seq, highly comprehensive candidate gene lists can be obtained from those profiling data [ 42 , 53 ].…”

“…Several factors need to be considered in the relative quantification, which include the quality and amount of mRNA, the efficiency of the reverse transcriptase, the primer amplification efficiency and the systematic and random variations [ 12 – 14 ]. Proper normalization is an important component of the precise measurement of mRNA and must deal with the cell count or the differences in tissue volume, the RNA concentration and purity variations, the efficiency of the reverse transcriptase and other amplification factors.…”

Development of a new formulation of interferons (HEBERPAG) for BCC treatment

“…Molecular characterization mRNA expression of genes potentially involved in the antitumoral effect of HEBERPAG was done using qPCR after incubation of cell lines with HEBERPAG during 72 h, in tissue culture conditions [19].…”

“…In xenograph mouse model it inhibits the tumor growth of Hep-2 cell line. Additionally, HEBERPAG down regulates expression of anti-apoptotic Bcl-2 and up-regulates apoptotic Bax, p53, and caspases mRNAs; and establishes a favorable antitumoral relationship between gene suppressor activity (STAT-1) and pro-oncogenic activity (STAT-3)[18][19][20].…”