Normalizing to GADPH jeopardises correct quantification of gene expression in ovarian tumours – IPO8 and RPL4 are reliable reference genes

Abstract

BackgroundTo ensure a correct interpretation of results obtained with quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), it is critical to normalize to a reference gene with stable mRNA expression in the tissue of interest. GADPH is widely used as a reference gene in ovarian tumour studies, although lacking tissue-specific stability. The aim of this study was to identify alternative suitable reference genes for RT-qPCR studies on benign, borderline, and malignant ovarian tumours.MethodsWe assayed mRNA levels for 13 potential reference genes – ABL1, ACTB, CDKN1A, GADPH, GUSB, HPRT1, HSP90AB, IPO8, PPIA, RPL30, RPL4, RPLPO, and TBP –with RT-qPCR in 42 primary ovarian tumours, using commercially pre-designed RT-qPCR probes. Expression stability was subsequently analysed with four different statistical programs (GeNorm, NormFinder, BestKeeper, and the Equivalence test).ResultsExpression of IPO8, RPL4, TBP, RPLPO, and ACTB had the least variation in expression across the tumour samples according to GeNorm, NormFinder, and BestKeeper. The Equivalence test found variation in expression within a 3-fold expression change between tumour groups for: IPO8, RPL40, RPL30, GUSB, TBP, RPLPO, ACTB, ABL1, and CDKN1A. However, only IPO8 satisfied at a 2-fold change as a cut-off. Overall, IPO8 and RPL4 had the highest, whereas GADPH and HPRT1 the lowest expression stability. Employment of suitable reference genes (IPO8, RPL4) in comparison with unsuitable ones (GADPH, HPRT1), demonstrated divergent influence on the mRNA expression pattern of our target genes − GPER and uPAR.ConclusionsWe found IPO8 and RPL4 to be suitable reference genes for normalization of target gene expression in benign, borderline, and malignant ovarian tumours. Moreover, IPO8 can be recommended as a single reference gene. Neither GADPH nor HPRT1 should be used as reference genes in studies on ovarian tumour tissue.

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

“…Interestingly, this result is consistent with the findings of Kolkova et al, where ABL1 turned out to be the least stable gene found by NormFinder analysis in human ovarian tumor tissue. 56 The combined analysis of 2D and 3D cultivated BMMSCs with NormFinder again determined not only GAPDH, but also GADD45A and RPL37A as the least stable RGs. In contrast, the DNA-damage-inducible gene GADD45A was identified as the most stable RG in a previous investigation of our group in osteogenically induced BM-MSCs (32).…”

Reference gene selection for real-time quantitative PCR analysis on ovarian cryopreservation by vitrification in mice

“…Traditionally, normalization is done using a single housekeeping gene [13]. However, several studies have revealed that these traditional reference genes are often not as stably expressed as expected, leading to misinterpretation of biological data [15,16]. Consequently, new and more stably expressed reference genes have been successfully identified in a variety of organisms [17], and the optimal reference genes were chosen to analyze the biological data accurately.…”

“…The enzymatic activity of PPIA may affect its stability as a reference gene. ACTB (actin, beta) is related to cell motility, structure, and integrity; it is a structural protein [16] and has good stability, likely because outside conditions do not generally affect cell structure. To track small changes in gene expression, use of single internal genes often does not produce accurate quantitative results, so two or more internal genes should be used for reference calibration.…”

“…ACTB is a cytoskeleton actin protein, and this kind of protein fiber network structure is very important in maintaining cell morphology; the influence of external factors is low [16,17]. GUSB, HPRT1, PPIA, and GAPDH are enzymes encoding [16,17] esters for TBP [16]; IPO8, UPAR, and GPER are receptors [17]; ABL1 is a receptor kinase [17]; and CDKN1A is a protease inhibitor [17]. Therefore, their stability is also different during different conditions, such as ovarian cryopreservation.…”

Genomic imbalances are involved in miR-30c and let-7a deregulation in ovarian tumors: implications for HMGA2 expression

“…Gene expression was assessed with Real-Time PCR using the TaqMan Gene Expression Assays (Applied Biosystems) for HMGA2 (Hs_04397751_m1), FHIT (Hs_00179987_m1), LIN28A (Hs_00702808_s1), and LIN28B (Hs_01013729_m1), whereas RPL4 (Hs_01939407_gH) was used as a reference gene as it has been proven to be stably expressed in ovarian cells [31, 32]. …”