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     

Stable reference genes for expression studies in breast muscle of normal and white striping-affected chickens

The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42 days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.

     

Gene Expression Studies in Formalin-Fixed Paraffin-Embedded Samples of Cutaneous Cancer: The Need for Reference Genes

Formalin-fixed paraffin-embedded (FFPE) tumour samples may provide crucial data regarding biomarkers for neoplasm progression. Analysis of gene expression is frequently used for this purpose. Therefore, mRNA expression needs to be normalized through comparison to reference genes. In this study, we establish which of the usually reported reference genes is the most reliable one in cutaneous malignant melanoma (MM) and cutaneous squamous cell carcinoma (CSCC). ACTB, TFRC, HPRT1 and TBP expression was quantified in 123 FFPE samples (74 MM and 49 CSCC biopsies) using qPCR. Expression stability was analysed by NormFinder and Bestkeeper softwares, and the direct comparison method between means and SD. The in-silico analysis with BestKeeper indicated that HPRT1 was more stable than ACTB and TFRC in MM (1.85 vs. 2.15) and CSCC tissues (2.09 vs. 2.33). The best option to NormFinder was ACTB gene (0.56) in MM and TFRC (0.26) in CSCC. The direct comparison method showed lower SD means of ACTB expression in MM (1.17) and TFRC expression in CSCC samples (1.00). When analysing the combination of two reference genes for improving stability, NormFinder indicated HPRT1 and ACTB to be the best for MM samples, and HPRT1 and TFRC genes for CSCC. In conclusion, HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM FFPE tissue, while the combination of HPRT1 and TFRC genes are the best option in analysing CSCC FFPE samples. These may be used consistently in forthcoming studies on gene expression in both tumours.     

Genome-Wide Association Study Identifies Loci and Candidate Genes for Body Composition and Meat Quality Traits in Beijing-You Chickens

Body composition and meat quality traits are important economic traits of chickens. The development of high-throughput genotyping platforms and relevant statistical methods have enabled genome-wide association studies in chickens. In order to identify molecular markers and candidate genes associated with body composition and meat quality traits, genome-wide association studies were conducted using the Illumina 60 K SNP Beadchip to genotype 724 Beijing-You chickens. For each bird, a total of 16 traits were measured, including carcass weight (CW), eviscerated weight (EW), dressing percentage, breast muscle weight (BrW) and percentage (BrP), thigh muscle weight and percentage, abdominal fat weight and percentage, dry matter and intramuscular fat contents of breast and thigh muscle, ultimate pH, and shear force of the pectoralis major muscle at 100 d of age. The SNPs that were significantly associated with the phenotypic traits were identified using both simple (GLM) and compressed mixed linear (MLM) models. For nine of ten body composition traits studied, SNPs showing genome wide significance (P<2.59E−6) have been identified. A consistent region on chicken (Gallus gallus) chromosome 4 (GGA4), including seven significant SNPs and four candidate genes (LCORL, LAP3, LDB2, TAPT1), were found to be associated with CW and EW. Another 0.65 Mb region on GGA3 for BrW and BrP was identified. After measuring the mRNA content in beast muscle for five genes located in this region, the changes in GJA1 expression were found to be consistent with that of breast muscle weight across development. It is highly possible that GJA1 is a functional gene for breast muscle development in chickens. For meat quality traits, several SNPs reaching suggestive association were identified and possible candidate genes with their functions were discussed.     

Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.     

The ability of genetically lean or fat slow-growing chickens to synthesize and store lipids is not altered by the dietary energy source

The increasing use of unconventional feedstuffs in chicken’s diets results in the substitution of starch by lipids as the main dietary energy source. To evaluate the responses of genetically fat or lean chickens to these diets, males of two experimental lines divergently selected for abdominal fat content were fed isocaloric, isonitrogenous diets with either high lipid (80 g/kg), high fiber (64 g/kg) contents (HL), or low lipid (20 g/kg), low fiber (21 g/kg) contents (LL) from 22 to 63 days of age. The diet had no effect on growth performance and did not affect body composition evaluated at 63 days of age. Glycolytic and oxidative energy metabolisms in the liver and glycogen storage in liver and Sartorius muscle at 63 days of age were greater in chicken fed LL diet compared with chicken fed HL diet. In Pectoralis major (PM) muscle, energy metabolisms and glycogen content were not different between diets. There were no dietary-associated differences in lipid contents of the liver, muscles and abdominal fat. However, the percentages of saturated (SFA) and monounsaturated fatty acids (MUFA) in tissue lipids were generally higher, whereas percentages of polyunsaturated fatty acids (PUFA) were lower for diet LL than for diet HL. The fat line had a greater feed intake and average daily gain, but gain to feed ratio was lower in that line compared with the lean line. Fat chickens were heavier than lean chickens at 63 days of age. Their carcass fatness was higher and their muscle yield was lower than those of lean chickens. The oxidative enzyme activities in the liver were lower in the fat line than in the lean line, but line did not affect energy metabolism in muscles. The hepatic glycogen content was not different between lines, whereas glycogen content and glycolytic potential were higher in the PM muscle of fat chickens compared with lean chickens. Lipid contents in the liver, muscles and abdominal fat did not differ between lines, but fat chickens stored less MUFA and more PUFA in abdominal fat and muscles than lean chickens. Except for the fatty acid composition of liver and abdominal fat, no interaction between line and diet was observed. In conclusion, the amount of lipids stored in muscles and fatty tissues by lean or fat chickens did not depend on the dietary energy source.     

Selection of valid reference genes for expression studies of hepatic cell lines under IFN-α treatment

The proper selection of reference genes to normalize the quantitative real-time PCR (RT-qPCR) results under particular experimental conditions is crucial for validation of the gene quantification data. Herein, using SYBR green RT-qPCR, five reference genes (GAPDH, ACTB, HMBS, HPRT-1 and TBP) were evaluated to determine the most stable reference genes in hepatic cell lines (Huh-7 and HepG₂) under IFN-α treatment conditions. Analyses by geNorm program ranked GAPDH and HPRT-1 in Huh-7 and that of ACTB and HMBS in HepG₂ cells as the most stable reference genes under IFN-α treatment. While, same reference gene pairs were ranked by NormFinder program in Huh-7 cells, GAPDH was assessed as the most stable gene in HepG₂ group by this program, implying the importance of the employed algorithm in comparative interpretation of the data. Finally, cumulative analyses by one-way ANOVA, geNorm and NormFinder programs indicated that use of two reference genes (HMBS and GAPDH) in Huh-7 and three (HMBS, ACTB and GAPDH) in HepG₂ cells would greatly improve the normalization of the RT-qPCR data under IFN-α. Data presented in this paper will aid the selection of the most stable reference genes in RT-qPCR studies on evaluation of hepatic viral proteins and IFN pathway.     

Mouse FKBP23 mediates conformer-specific functions of BiP by catalyzing Pro117 cis/trans isomerization

Gene expression analysis is frequently used to analyze the response to viral infection, and 18S RNA, SHDA and GAPDH represent popular house keeping genes (HKGs) often used to normalize gene expression. Here we describe the first systematic selection and evaluation of suitable HKGs for gene expression analysis in chicken embryo fibroblasts (CEF) infected with NDV adapted to the guidelines from Gorzelniak and Ferguson. Our results indicate that ACTB, HPRT1 and HMBS were valuable and stable HKGs, while 18S RNA, GAPDH and SHDA are considerably regulated during the course of infection and thus precluded for normalization. Normalizing the infection dependent gene IFN-a and the infection independent gene B2M to inappropriate HKGs consequently misleads to significant errors in estimating their regulations. Our study emphasizes that even the most popular HKGs like 18S RNA and GAPDH can lead to divergent and inaccurate data interpretation of significant magnitude if not carefully analyzed for stability before.     

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.     

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.     

Selection of reference genes for gene expression studies in astrocytomas

This study was aimed to test a panel of six housekeeping genes (GAPDH, HPRT1, POLR2A, RPLP0, ACTB, and H3F) so as to identify and validate the most suitable reference genes for expression studies in astrocytomas. GAPDH was the most stable and HPRT1 was the least stable reference gene. The effect of reference gene selection on quantitative real-time polymerase chain reaction data interpretation was demonstrated, normalizing the expression data of a selected gene of interest. Thus, GAPDH may be recommended for data normalization in gene expression studies in astrocytomas. Nevertheless, a preliminary validation of reference gene stability is required prior to every study.     

Analysis of synonymous codon usage patterns of HPRT1 gene across twelve mammalian species

Genetic changes in Hypoxanthine guanine phosphoribosyltransferace (HPRT1) gene can alter the expression of the dopamine neurotransmitter leads to abnormal neuron function, a disease called Lesch-Nyhan syndrome (LNS). Although different studies were conducted on LNS, information on codon usage bias (CUB) of HPRT1 gene is limited. The present study examines the genetic determinants of CUB in HPRT1 gene using twelve mammalian species. In the coding sequence of HPRT1 genes, A/T ending codons was most frequently used. A higher ENC value was observed indicating lower HPRT1 gene expression in the selected mammalian species. Correlation analysis indicates that compositional constraints under mutation pressure can involve in CUB of HPRT1 genes among the selected mammalian species. Relative synonymous codon usage (RSCU) value revealed that the codons such as ACT, AGG, ATT and AGC were over-represented in each of the mammalian species. Result from the analysis of the RSCU indicates that compositional constraint is a key driver for the variation in codon usage. Ratio of nonsynonymous (dN) and synonymous (dS) substitution further suggested that purifying selection occurs among the HPRT1 gene of studied mammals to maintain its protein function under the process of evolution. Our findings report an insight into the codon usage patterns of HPRT1 gene and will be useful for LNS management.     

Reference Genes Selection for Quantitative Real-Time PCR Using RankAggreg Method in Different Tissues of Capra hircus

Identification of reference genes with stable levels of gene expression is an important prerequisite for obtaining reliable results in analysis of gene expression data using quantitative real time PCR (RT-qPCR). Since the underlying assumption of reference genes is that expressed at the exact same level in all sample types, in this study, we evaluated the expression stability of nine most commonly used endogenous controls (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, and B2M) in four different tissues of the domestic goat, Capra hircus, including liver, visceral, subcutaneous fat and longissimus muscles, across different experimental treatments (a standard diet prepared using the NRC computer software as control and the same diet plus one mg chromium/day). We used six different software programs for ranking of reference genes and found that individual rankings of the genes differed among them. Additionally, there was a significant difference in ranking patterns of the studied genes among different tissues. A rank aggregation method was applied to combine the ranking lists of the six programs to a consensus ranking. Our results revealed that HSP-90 was nearly always among the two most stable genes in all studied tissues. Therefore, it is recommended for accurate normalization of RT-qPCR data in goats, while GAPDH, ACTB, and RPS18 showed the most varied expressions and should be avoided as reference genes.