Identification of suitable reference genes for gene expression studies of human serous ovarian cancer by real-time polymerase chain reaction

Quantitative real-time RT-PCR (RT-qPCR) has proven to be a valuable molecular technique in gene expression quantification. Target gene expression levels are usually normalized to a stably expressed reference gene simultaneously determined in the same sample. It is critical to select optimal reference genes to interpret data generated by RT-qPCR. However, no suitable reference genes have been identified in human ovarian cancer to date. In this study, 10 housekeeping genes, ACTB, ALAS1, GAPDH, GUSB, HPRT1, PBGD, PPIA, PUM1, RPL29, and TBP as well as 18S rRNA that were already used in various studies were analyzed to determine their applicability. Totally 20 serous ovarian cancer specimens and 20 normal ovarian epithelial tissue specimens were examined. All candidate reference genes showed significant differences in expression between malignant and nonmalignant groups except GUSB, PPIA, and TBP. The expression stability and suitability of the 11 genes were validated employing geNorm and NormFinder. GUSB, PPIA, and TBP were demonstrated as the most stable reference genes and thus could be used as reference genes for normalization in gene profiling studies of serous ovarian cancer, while the combination of two genes (GUSB and PPIA) or the all three genes should be recommended as a much more reliable normalization strategy.     

小鼠基因转录表达分析中内参基因的优选

目的 建立小鼠基因转录表达分析中内参基因的选择方法.方法 以C57BL/6J和C3H/HeJ两个品系3个不同组织及2个不同发育阶段为研究对象,应用反转录实时定量PCR技术,评价GAPDH(glyceraldehyde-3-phosphate dehydrogenase)、HPRTl(hypoxanthine phosphoribosyl transferase)、B2M(β2-microglobulin)、PPIA(peptidylprolyl isomerase A)、ACTB(Actin-beta)和18S rRNA(18S ribosomal RNA)等6个看家基因在下丘脑、垂体与卵巢中mRNA水平的表达稳定性.结果 GeNorm统计分析表明,GAPDH和HPRT1表达最为稳定,PPIA等次之,B2M在不同组织和发育阶段中都几乎无表达.结论 成功筛选到GAPDH和HPRT1两个稳定表达的看家基因,证实了小鼠基因表达转录分析中内参基因选择的必要性和可行性.     

Normalizing genes for real-time polymerase chain reaction in epithelial and nonepithelial cells of mouse small intestine

Gene expression studies in intestinal epithelial and stromal cells are a common tool for investigating the mechanisms by which the homeostasis of the small intestine is regulated under normal and pathological conditions. Quantitative real-time PCR (qPCR) is a sensitive and highly reproducible method of gene expression analysis, with expression levels quantified by normalization against reference genes in most cases. However, the lack of suitable reference genes for epithelial cells with different differentiation states and nonepithelial tissue cells has limited the application of qPCR in gene expression studies of small intestinal samples. In this study, 13 housekeeping genes, ACTB, B2M, GAPDH, GUSB, HPRT1, HMBS, HSP90AB1, RPL13A, RPS29, RPLP0,PPIA, TBP, and TUBA1, were analyzed to determine their applicability for isolated crypt cells, villus cells, deepithelialized mucosa, and whole mucosa of the mouse small intestine. Using geNorm and NormFinder software, GUSB and TBP were identified as the most stably expressed genes, whereas the expressions of the commonly used reference genes GAPDH, B2M, and ACTB, and ribosomal protein genes RPL13A, RPS29, and RPLP0 were relatively unstable. Thus, this study demonstrates that GUSB and TBP are the optimal reference genes for the normalization of gene expression in the mouse small intestine.     

The Use of Laser Microdissection in the Identification of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Human FFPE Epithelial Ovarian Tissue Samples

Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.     

Identifying suitable reference genes for developing and injured mouse CNS tissues

Accurate quantification of gene expression is fundamental for understanding the molecular, genetic and functional bases of tissue development and diseases. Quantitative real‐time PCR (qPCR) is now the most widely used method of quantifying gene expression due to its simplicity, specificity, sensitivity, and wide quantification range. The use of appropriate reference genes to ensure accurate normalization is crucial for the correct quantification of gene expression from the early development, maturation, aging to injury processes in the central nervous system (CNS). In this study, we have determined the expression profiles of 12 candidate housekeeping genes (ACTB, CYC1, HMBS, GAPDH, HPRT1, RPL13A, YWHAZ, PPIA, RPLP0, TFRC, GUS, and 18S rRNA) in developing mouse brain and spinal cord. Throughout development, there was a significant degree of fluctuations in their expression levels, indicating the importance and complexity of finding appropriate reference genes. Three software including BestKeeper, geNorm and NormFinder were used to evaluate the stability of potential reference genes. GUS was the most stable gene and GUS/YWHAZ were the most stable reference gene pair across different developmental stages in different CNS regions, whereas HPRT1 and GAPDH were the most variable genes and thus inappropriate to use as reference genes. Therefore, our results identified GUS and YWHAZ as the best combination of two reference genes for expression data normalization in CNS developmental studies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 39–50, 2018     

Evaluation of reference genes for quantitative real-time PCR in the brain,pituitary, and gonads of songbirds

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.     

Evaluating reference genes to normalize gene expression in human epileptogenic brain tissues

Several reference genes have been used to quantify gene expression in human epilepsy surgery tissue. However, their reliability has not been validated in detail, although this is crucial in interpreting epilepsy-related changes of gene expression. We evaluated 12 potential reference genes in neocortical tissues resected from patients with temporal lobe epilepsy (TLE) with either few or many seizures (n=6 each) and post mortem controls (n=6) using geNorm and NormFinder algorithms. For all candidate reference genes threshold cycle (C(T)) values were measured. geNorm analysis revealed that the expression of e.g. glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl-transferase (HPRT) is unstable, whereas synaptophysin (SYP) and neuron-specific enolase (NSE)/mitochondrial 39S ribosomal protein L28 (MRPL) are most stably expressed. The geometric mean of SYP, NSE and MRPL levels is recommended as normalization factor (NF). NormFinder analysis, in contrast, indicated HPRT as the most stable single gene and recommended the geometric mean of TATA-box binding protein (TBP) and NSE levels as NF. Different values of upregulation of glial fibrillary protein (GFAP) expression were found in TLE tissue compared to control tissue depending on the NF used: 4.5-fold (geNorm-NF), 4.7-fold (NormFinder-NF), 4.2-fold (vs. GAPDH) and 7.8-fold (vs. HPRT). The expression of GABA(A) receptor subunit α5 (GARα5) was unaltered in the TLE groups compared to controls (geNorm-NF, NormFinder-NF, vs. GAPDH). However, normalization to HPRT suggests an apparent increase of GARα5 expression. In conclusion, the geNorm-NF (SYP/NSE/MRPL) and the NormFinder-NF (TBP/NSE) are equally suitable for normalization of gene expression in the human epileptogenic neocortex. In contrast, normalization to single and probably less stably expressed genes may not deliver accurate results.     

Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation

Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase^plus software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results     

光裸星虫(Sipunculus nudus)不同发育时期卵细胞内参基因的筛选

内参基因的选择对功能基因表达量的归一化处理尤为重要。为了筛选出光裸星虫不同发育时期卵子的最适内参基因,利用qRT-PCR测定了甘油醛-3-磷酸脱氢酶(GAPDH)、肽基脯氨酰顺反异构酶A(PPIA)、60S核糖体蛋白L10(60S-L10)、铁蛋白(Ferritin)、β-肌动蛋白(β-actin)、泛素C(UBC)、真核生物翻译起始因子(eIF)、NADH脱氢酶(NDH)、28S核糖体RNA(28S)、TATA盒结合蛋白(TBP)、18S核糖体RNA(18S)和琥珀酸脱氢酶A亚基(SDHA)共12个候选内参基因的表达水平,并通过4个程序(geNorm,NormFinder,BestKeeper以及RefFinder)综合分析了各基因的表达稳定性。结果显示:(1)12个候选内参基因均能获得特异性扩增产物,但表达情况各异;(2)对候选内参基因进行综合打分,得到候选内参基因稳定性排名为18S>GAPDH>28S>β-actin>UBC>e IF>NDH|TBP>PPIA|Ferritin>60S-L10>SDHA。18S和GAPDH稳定性较好,可作为不同发育时期卵细胞基因表达研究的单内参基因,或最优组合内参基因。     

Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data

Real-time PCR is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields. The significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. The most widely used approach is to use endogenous reference genes (housekeeping genes) as internal standards. This approach is controversially discussed in the literature because none of the reference genes is stably expressed throughout all biological samples. Therefore, candidate reference genes have to be validated for each experimental condition. In our studies, we introduced and evaluated an in vitro synthesized reference cRNA for internal standardization of relative messenger RNA (mRNA) expression patterns. This reference, consisting of the in vitro transcribed coding sequence of aequorin, a jellyfish protein, was incorporated in the extracted RNA. The experimental significance of this approach was representatively tested for the expression of the neurotrophin-3 mRNA in distinct regions of mouse brains. A comparison to three stably expressed reference genes [beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyl-transferase 1 (HPRT1)] gave evidence that the spiking of template RNA with in vitro transcribed cRNA is a valuable tool for internal standardization of real-time PCR experiments.     

Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression

Real-time PCR is the benchmark method for measuring mRNA expression levels, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. Though the minipig model is largely used to study cardiovascular disease, no specific reference genes have been identified in porcine myocardium. The aim of the study was to identify and validate reference gene to be used in RT-PCR studies of failing (HF) and non-failing pig hearts. Eight candidate reference genes (GAPDH, ACTB, B2M, TBP, HPRT-1, PPIA, TOP2B, YWHAZ) were selected to compare cardiac tissue of normal (n=4) and HF (n=5) minipigs. The most stable genes resulted: HPRT-1, TBP, PPIA (right and left atrium); PPIA, GAPDH, ACTB (right ventricle); HPRT-1, TBP, GAPDH (left ventricle). The normalization strategy was tested analyzing mRNA expression of TNF-α, which is known to be up-regulated in HF and whose variations resulted more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance to provide a set of reference genes to normalize mRNA expression in HF and control minipigs. The use of unvalidated reference genes can generate biased results because also their expression could be altered by the experimental conditions.     

Evidence based selection of housekeeping genes

For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes (reference or internal control genes) is required. It is known that commonly used housekeeping genes (e.g. ACTB, GAPDH, HPRT1, and B2M) vary considerably under different experimental conditions and therefore their use for normalization is limited. We performed a meta-analysis of 13,629 human gene array samples in order to identify the most stable expressed genes. Here we show novel candidate housekeeping genes (e.g. RPS13, RPL27, RPS20 and OAZ1) with enhanced stability among a multitude of different cell types and varying experimental conditions. None of the commonly used housekeeping genes were present in the top 50 of the most stable expressed genes. In addition, using 2,543 diverse mouse gene array samples we were able to confirm the enhanced stability of the candidate novel housekeeping genes in another mammalian species. Therefore, the identified novel candidate housekeeping genes seem to be the most appropriate choice for normalizing gene expression data.     

Reference Gene Selection for Real-Time Quantitative PCR Analysis of the Mouse Uterus in the Peri-Implantation Period

The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm^PLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.     

Novel reference gene RPN1 for normalization of quantitative data in lung and kidney cancer

Quantitative methods of gene expression analysis in tumors require accurate data normalization, which allows comparison of different mRNA/cDNA samples with unknown concentration. For this purpose reference genes with stable expression level (such as GAPDH, ACTB, HPRT1, TBP) are used. The choice of appropriate reference genes is still actual because well-known reference genes are not suitable for certain cancer types frequently and their unreasonable use without additional tests lead to wrong conclusions. We have developed the bioinformatic approach and selected a new potential reference gene RPN1 for lung and kidney tumors. This gene is located at the long arm of chromosome 3. Our method includes mining of the dbEST and Oncomine databases and functional analysis of genes. The RPN1 was selected from 1500 candidate housekeeping genes. Using comparative genomic hybridization with NotI-microarrays we found no methylation, deletions and/or amplifications at the RPN1-containing locus in 56 non-small cell lung and 42 clear cell renal cancer samples. Using RT-qPCR we showed low variability of RPN1 mRNA level comparable to those of reference genes GAPDH and GUSB in lung and kidney cancer. The mRNA levels of two target genes coding hyalouronidases--HYAL1 and HYAL2--were estimated and normalized relative to pair RPN1--GAPDH genes for lung cancer and RPN1--GUSB for kidney cancer. These combinations were shown to be optimal for obtaining accurate and reproducible data. All obtained results allow us to suggest RPN1 as novel reference gene for quantitative data normalization in gene expression studies for lung and kidney cancers.     

Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes

Quantitative real-time RT-PCR was used to investigate the effects of prototypical drug-metabolizing enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on mRNA expression levels of the housekeeping genes beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolylisomerase A (PPIA), TATA box binding protein (TBP), and transferrin receptor (TFRC) in primary cultures of cryopreserved human and rat hepatocytes. The mRNA levels of ACTB, GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in human hepatocytes were constant at all concentrations of inducers. However, the mRNA level of GAPDH relative to HPRT1 in rat hepatocytes was markedly increased by Rif. The mRNA levels of GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in rat hepatocytes were significantly increased by Dex. ACTB and HPRT1 are suitable internal controls for evaluating mRNA expression levels in primary cultures of human and rat hepatocytes after Rif, Dex, or Ome exposure.     

Selection of Reference Genes for qRT-PCR in High Fat Diet-Induced Hepatic Steatosis Mice Model

With the epidemic proportions of obesity worldwide and the concurrent prevalence of hepatic steatosis, there is an urgent need for better understanding the intrinsic mechanism of hepatic steatosis, especially the changes of gene expression underlying the development of hepatic steatosis and its associated abnormal liver function. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible technique of gene expression analysis. However, for accurate and reliable gene expression results, it is vital to have an internal control gene expressed at constant levels under all the experimental conditions being analyzed for. In this study, the authors validated candidate reference genes suitable for qRT-PCR profiling experiments using livers from control mice and high fat diet-induced obese mice. Cross-validation of expression stability of ten selected reference genes using three popular algorithms, GeNorm, NormFinder, and BestKeeper found HPRT1 and GAPDH as most stable reference genes. Thus, HPRT1 and GAPDH are recommended as stable reference genes most suitable for gene expression studies in the development of hepatic steatosis.     

Effects of dimethyl sulfoxide and dexamethasone on mRNA expression of housekeeping genes in cultures of C2C12 myotubes

We used quantitative real-time RT-PCR to investigate the effects of dimethyl sulfoxide (DMSO) and dexamethasone (Dex) on the mRNA expression levels of the housekeeping genes β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), phosphoglycerate kinase 1 (PGK1), peptidylprolyl isomerase A (PPIA), and transferrin receptor (TFRC) in cultures of C2C12 myotubes. The ratios of ACTB mRNA levels to the HPRT1 mRNA level in C2C12 cells that were differentiating from myoblast cells to myotubes decreased from 0 to 120 h of culture, whereas the ratios of TFRC mRNA levels to the HPRT1 mRNA level increased from 0 to 120 h of culture. The ratios of GAPDH, GUSB, PGK1, and PPIA mRNA levels to the HPRT1 mRNA level remained constant from 0 to 120 h of culture. All housekeeping gene mRNA levels were unaffected by exposure to DMSO concentrations of 0.1% or less. The GAPDH mRNA level was increased by Dex, while the ACTB and PGK1 mRNA levels were significantly decreased by Dex. The GUSB, PPIA, and TFRC mRNA levels were unaffected by exposure to Dex. GUSB, HPRT1, and PPIA are thus suitable internal controls for evaluating mRNA expression levels in cultures of C2C12 cells.