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

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), ß-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.

High Functional Stability of a Low-cost HBV DNA qPCR Primer Pair and Plasmid Standard.

AIM: We studied the functional stability of a primer pair and the standard curve based on a plasmid carrying full-length HBV genome, from a novel low-cost real-time quantitative polymerase chain reaction (qPCR) assay. The assay was developed at the Center for Genetic Engineering and Biotechnology (CIGB) in Havana, to quantify the serum hepatitis B virus (HBV) DNA from chronic HBV-infected (CHB) patients. MATERIALS AND METHODS: In-house generated oligonucleotides and plasmids were incubated at 37°C during 1 month and compared with the same materials incubated at -20, 4, and 25°C during the same time in qPCR experiments. RESULTS: This work shows that the oligonucleotide pair and the plasmid for the quantitative standard curve are functionally stable in severe temperature conditions during 1 month. Polymerase chain reaction amplification with both materials after its incubation 30 days at 37°C produced similar cycle threshold (CT) values and similar degree of sample quantifications compared with the same materials preserved using the conventional storage conditions at -20°C. CONCLUSION: These results are indicative of the robustness of this low-cost qPCR system for HBV DNA quantification. These results also support that this qPCR assay can be used as a low-cost technology in clinical studies to monitor the viral load changes of serum HBV DNA of CHB patients, which could be used by poor people of third world countries, where there are frequent blackouts and temperature changes that can hinder the primer and plasmid stability. HOW TO CITE THIS ARTICLE: Aguiar J, García G, León Y, Canales E, Silva JA, Gell O, Estrada R, Morán I, Muzio V, Guillén G, Pentón E, Aguilar JC. High Functional Stability of a Low-cost HBV DNA qPCR Primer Pair and Plasmid Standard. Euroasian J Hepato-Gastroenterol 2016;6(1):19-24.