Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus.

The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.).

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

Evaluation of reference genes for quantitative expression analysis in Mylabris sibirica (Coleoptera, Meloidae).

Mylabris sibirica is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in Mylabris sibirica, have been tremendously limited. In this study, ten internal reference genes (ACT, ARF1, AK, EF1α, GAPDH, α-TUB, RPL6, RPL13, RPS3 and RPS18) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that RPL6 and RPL13 were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of RPL6 and RPS18 was recommended to test gene transcription levels under different adult tissues. AK and RPL6 were the best reference genes in male and female adults. RPL6 and RPL13 were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase (MsUAP), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in M. sibirica, such as development, reproduction, sex differentiation, cold and heat resistance.

Housekeeping gene stability in Pesudomonas aeruginosa PAO1 under the pressure of commonly used antibiotics in molecular microbiology assays.

Pseudomonas aeruginosa is an opportunistic human pathogen notorious for its remarkable capacity of multi-drug resistance, and has become one of the most important model bacteria in clinical bacteriology research. Quantitative real-time PCR is a reliable method widely used in gene expression analysis, for which the selection of a set of appropriate housekeeping genes is a key prerequisite for the accuracy of the results. However, it is easy to overlook that the expression level of housekeeping gene may vary in different conditions, especially in the condition of molecular microbiology assays, where tested strains are generally cultured under the pre-set antibiotic selection pressures, and how this affects the stability of commonly used housekeeping genes remains unclear. In this study, the expression stability of ten classic housekeeping genes (algD, gyrA, anr, nadB, recA, fabD, proC, ampC, rpoS, and rpsL) under the pressure of eight laboratory commonly used antibiotics (kanamycin, gentamycin, tetracycline, chloramphenicol, hygromycin B, apramycin, tellurite, and zeocin) were tested. Results showed that the stability of housekeeping gene expression was indeed affected by the types of antibiotics added, and of course the best reference gene set varied for different antibiotics. This study provides a comprehensive summary of the effects of laboratory antibiotics on the stability of housekeeping genes in P. aeruginosa, highlighting the necessity to select housekeeping genes according to the type of antibiotics used in the initial stage of experiment.

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata.

Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai-Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata. In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS, EF-1α, and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP, TFIIS, and CYP22 were the optimal reference genes in the roots of L. rotata. The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats.

Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives.

Lettuce is a popular vegetable source of bioactive compounds, like anthocyanins, powerful antioxidants present in red and semi-red varieties. Selection of reliable reference genes (RGs) for the normalization of real-time quantitative PCR (qPCR) data is crucial to obtain accurate gene expression results. Among the genes with totally unrelated biological functions, six candidate RGs (ADF2, CYB5, iPGAM, SCL13, TRXL3-3, and VHA-H) with low variation in expression according to RNA-seq analyses, were selected for future expression studies of anthocyanin-related genes in three different experiments: leaf colour comparison (green vs. red) in commercial varieties; tissue comparison (leaf vs. stem) in a wild relative; and drought stress experiment in commercial and traditional varieties, and a wild relative. Expression profiles of the candidate RGs were obtained by qPCR and their stability was assessed by four different analytical tools, geNorm, NormFinder, BestKeeper, and Delta Ct method, all integrated in RefFinder. All results considered, we recommend CYB5 to be used as RG for the leaf colour experiment and TRXL3-3 for the tissue and drought stress ones, as they were the most stable genes in each case. RNA-seq is useful to preselect novel RGs although validation by qPCR is still advisable. These results provide helpful information for gene expression studies in Lactuca spp. under the described conditions.

Selection and validation of appropriate reference genes for RT-qPCR analysis of Nitraria sibirica under various abiotic stresses.

BACKGROUND: Nitraria sibirica Pall. is a halophytic shrub with strong environmental adaptability that can survive in extremely saline-alkali and drought-impacted environments. Gene expression analysis aids in the exploration of the molecular mechanisms of plant responses to abiotic stresses. RT-qPCR is the most common technique for studying gene expression. Stable reference genes are a prerequisite for obtaining accurate target gene expression results in RT-qPCR analysis. RESULTS: In this study, a total of 10 candidate reference genes were selected from the transcriptome of N. sibirica, and their expression stability in leaves and roots under different treatment conditions (salt, alkali, drought, cold, heat and ABA) was evaluated with the geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the tissue and experimental conditions tested. ACT7 combined with R3H, GAPDH, TUB or His were the most stable reference genes in the salt- or alkali-treated leaves, salt-treated roots and drought-treated roots, respectively; R3H and GAPDH were the most suitable combination for drought-treated leaves, heat-treated root samples and ABA-treated leaves; DIM1 and His maintained stable expression in roots under alkali stress; and TUB combined with R3H was stable in ABA-treated roots. TBCB and GAPDH exhibited stable expression in heat-treated leaves; TBCB, R3H, and ERF3A were stable in cold-treated leaves; and the three most stable reference genes for cold-treated roots were TBCB, ACT11 and DIM1. The reliability of the selected reference genes was further confirmed by evaluating the expression patterns of the NsP5CS gene under the six treatment conditions. CONCLUSION: This study provides a theoretical reference for N. sibirica gene expression standardization and quantification under various abiotic stress conditions and will help to reveal the molecular mechanisms that confer stress tolerance to N. sibirica.

Reference Gene Validation for RT-qPCR in PBMCs from Asthmatic Patients with or without Obesity.

Obesity is known to impair the efficacy of glucocorticoid medications for asthma control. Glucocorticoid-induced gene expression studies may be useful to discriminate those obese asthmatic patients who present a poor response to glucocorticoids. The expression of genes of interest is normalized with respect to reference genes (RGs). Ideally, RGs have a stable expression in different samples and are not affected by experimental conditions. The objective of this work was to analyze suitable RGs to study the role of glucocorticoid-induced genes in obese asthmatic patients in further research. The gene expression of eight potential RGs (GUSB, B2M, POLR2A, PPIA, ACTB, GAPDH, HPRT1, and TBP) was assessed with reverse transcription-quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from asthmatic, obese asthmatic, and healthy individuals. Their stability was analyzed using four different algorithms-BestKeeper, ΔCt, geNorm, and NormFinder. geNorm analysis recommended the use of a minimum of three genes for normalization. Moreover, intergroup variation due to the treatment was calculated by NormFinder, which found that B2M was the gene that was least affected by different treatments. Comprehensive rankings indicated GUSB and HPRT1 as the best RGs for qPCR in PBMCs from healthy and asthmatic subjects, while B2M and PPIA were the best for obese asthmatic subjects. Finally, our results demonstrated that B2M and HPRT1 were the most stable RGs among all groups, whereas ACTB, TBP, and GAPDH were the worst shared ones.

TBP, PPIA, YWHAZ and EF1A1 Are the Most Stably Expressed Genes during Osteogenic Differentiation.

RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes' expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases.

Identification and validation of stage-specific reference genes for gene expression analysis in Callosobruchus maculatus (Coleoptera: Bruchidae).

In light of a number of recent studies highlighting the increasing research interest in bruchids, it is crucial to validate suitable reference genes that could be used in quantitative gene expression studies. Callosobruchus maculatus is a serious pest of stored grains and field legumes in which reference genes have not been assessed and validated to date. The present study aimed to identify and validate reference genes in different developmental stages of C. maculatus shortlisted from commonly used reference genes such as VATPase, TRIP12, TBP, TF11D, ACTIN, GST, ANNEXIN, PTCD3, RPL32, and ß -Tub in various insects. Dedicated algorithms like GeNorm, NormFinder, and BestKeeper were used to analyze the stability of these candidate genes, which revealed GST for third instar, ANNEXIN and PTCD3 for the fourth instar, TF11D and VATPase for male pupa, RPL32 and ß-tub for female pupa, ß-tub and TBP for adult male and VATPase and GST for adult females as suitable reference genes for expression studies in C. maculatus. The final comprehensive ranking using RefFinder identified GST and TBP as the best reference genes for all the developmental stages of C. maculatus. To the best of our knowledge, this is the first report which evaluates and validates stable reference genes in C. maculatus. The information of stage-specific gene expression, generated in this study will be useful for future molecular, physiological, and biochemical studies on C. maculatus and other closely related bruchids.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum.

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

Selection of suitable internal controls for gene expression normalization in rats with spinal cord injury.

There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research. In this study, we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes: Actb, Ankrd27, CypA, Gapdh, Hprt1, Mrpl10, Pgk1, Rictor, Rn18s, Tbp, Ubc, Ubxn11, and Ywhaz. Our results show that the expression of Ankrd27, Ubc, and Tbp were stable after spinal cord injury, while Actb was the most unstable internal control gene. Ankrd27, Ubc, Tbp, and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression. Target gene expression levels and changes over time were similar when Ankrd27, Ubc, and Tbp were used as internal controls but different when Actb was used as an internal control. We recommend that Ankrd27, Ubc, and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research. This study was approved by the Administration Committee of Experimental Animals, Jiangsu Province, China (approval No. 20180304-008) on March 4, 2018.

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries.

BACKGROUND: Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. RESULTS: We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. CONCLUSIONS: We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress.

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI, dnaG, and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytvI, dnaG, and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.

Selection of Suitable Reference Genes for qRT-PCR Gene Expression Studies in Rice.

With a widely established use of quantitative real-time PCR (qRT-PCR) for gene expression analysis, reliable and stable expression of reference genes is often discussed. Suitable reference genes should show less variation of expression across the target samples and allow for error minimization by normalization of qRT-PCR data. Therefore, selection of reliable reference genes is essential for accurate results and to support the conclusions drawn on expression levels of genes under study. In this chapter, we describe the workflow for selection and evaluation of reference genes in rice, including identification of candidate genes by using Genevestigator® and evaluation of expression stability using various algorithms. The ranking of the genes guides qRT-PCR performance and data analysis. This protocol used rice as an example but is not limited to rice, and could be applied to other species as well.

Identification and validation of suitable reference genes for quantitative real-time PCR gene expression analysis in pregnant human myometrium.

Accurate quantification of quantitative PCR (qPCR) data requires a set of stable reference genes (RGs) for normalisation. Despite its importance to mechanistic studies, no evaluation of RG stability has been conducted for pregnant human myometrium. A systematic search of the literature was performed to identify the most used RGs in human myometrial gene expression studies. The stability of these genes, and others, was then evaluated using geNorm and NormFinder algorithms, in samples of myometrium from singleton or twin pregnancies (n = 7 per group) delivering at term or preterm. The most frequently cited RGs were GAPDH, ACTB, B2M and 18s. There was strong agreement between algorithms on the most and least stable genes: Both indicated CYC1, YWHAZ and ATP5B were the most stably expressed. Despite being some of the most used RGs, B2M, 18s and ACTB expression was least stable and was too variable for use as accurate normalisation factors. Pairwise variation analysis determined that the optimal number of RGs for accurate normalisation is two. Validation of the choice of RGs by comparing relative expression of oxytocin receptors (OXTR) using the least stable 18s and B2M, with the most stable, CYC1 and YWHAZ, erroneously demonstrated significantly increased OXTR expression in myometrium in singleton pregnancies compared to twins. This study demonstrates the importance of appropriate RG selection for accurate quantification of relative expression in pregnant human myometrium qPCR studies. For normalisation, the geometric mean of CYC1 and YWHAZ or ATP5B is suggested. The use of ACTB, 18s and B2M, is not recommended.

Identification and validation of reference genes for qRT-PCR based studies in horse gram (Macrotyloma uniflorum).

The quantitative real-time polymerase chain reaction (qRT-PCR) is the most sensitive and commonly used technique for gene expression studies in biological systems. However, the reliability of qRT-PCR results depends on the selection of reference gene(s) for data normalization. Horse gram (Macrotyloma uniflorum) is an important legume crop on which several molecular studies have been reported. However, the stability of reference genes has not been evaluated. In the present study, nine candidate reference genes were identified from horse gram RNA-seq data and evaluated in two horse gram genotypes, HPK4 and HPKM317 under six abiotic stresses viz. cold, drought, salinity, heat, abscisic acid and methyl viologen-induced oxidative stress. The results were evaluated using geNorm, Bestkeeper, Normfinder and delta-delta Ct methods and comprehensive ranking was assigned using RefFinder and RankAggreg software. The overall result showed that TCTP was one of the most stable genes in all samples and in genotype HPK4, while in HPKM317 profilin was most stably expressed. However, PSMA5 was identified as least stable in all the experimental conditions. Expression of target genes dehydrin and early response to dehydration 6 under drought stress was also validated using TCTP and profilin for data normalization, either alone or in combination, which confirmed their suitability for qRT-PCR data normalization. Thus, TCTP and profilin genes may be used for qRT-PCR data normalization for molecular and genomic studies in horse gram. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01104-0.

A new set of reference genes for comparative gene expression analyses in Yarrowia lipolytica.

Accurate quantitation of gene expression levels require sensitive, precise and reproducible measurements of specific transcripts. Normalization to a reference gene is the most common practice to minimize the impact of the uncontrolled variation. The fundamental prerequisite for an accurate reference gene is to be stably expressed amongst all the samples included in the analysis. In the present study we aimed to assess the expression level and stability of a panel of 21 genes in Yarrowia lipolytica throughout varying conditions, covering composition of the culturing medium, growth phase and strain-wild type and recombinant burdened with heterologous protein overexpression. The panel of the selected candidate genes covered those essential for growth and maintenance of metabolism and homologs of commonly used internal references in RT-qPCR. The candidate genes expression level and stability were assessed and the data were processed using dedicated computational tools (geNorm and NormFinder). The results obtained here indicated genes unaffected by the burden of overexpression (TEF1, TPI1, UBC2, SRPN2, ALG9-like, RYL1) or by the culture medium used (ACT1, TPI1, UBC2, SEC61, ODC, CLA4, FKS1, TPS1), as well as those the least (SSDH, ODC, GPD) and the most (SEC62, TPI1, IPP1) suitable for normalization of RT-qPCR data in Y. lipolytica.

Using RNA-Seq to Identify Reference Genes of the Transition from Brown to White Adipose Tissue in Goats.

Brown adipose tissues have unique non-shivering thermogenesis functions, can be found in newborn ruminate animals, and then are gradually replaced by white adipose tissues in adulthood. For the purpose of exploring the intrinsic mechanism underlying the conversion process from brown (BAT) to white adipose tissue (WAT), it is necessary to utilize Quantitative PCR (qPCR) to study gene expression profiling. In this study, we identified reference genes that were consistently expressed during the transformation from goat BAT to WAT using RNA-seq data. Then, twelve genes were evaluated as candidate reference genes for qPCR in goat perirenal adipose tissue using three tools (geNorm, Normfinder, and BestKeeper). In addition, the selected reference genes were used to normalize the gene expression of PGC-1α and GPAT4. It was found that traditional reference genes, such as GAPDH, RPLP0, HPRT1, and PPIA were not suitable for target gene normalization. In contrast, CTNNB, PFDN5, and EIF3M, selected from RNA sequencing data, showed the least variation and were recommended as the best reference genes during the transformation from BAT to WAT.

Whole blood ACTB, B2M and GAPDH expression reflects activity of inflammatory bowel disease, advancement of colorectal cancer, and correlates with circulating inflammatory and angiogenic factors: Relevance for real-time quantitative PCR.

BACKGROUND: The effect of bowel inflammation and cancer on the expression of the most prevalent internal controls: ACTB, GAPDH and B2M in whole blood is unknown, although at least GAPDH occurred to be tightly regulated and suspected of supporting cancer growth, challenging its suitability as a reference. OBJECTIVES: To evaluate the effect of colorectal cancer (CRC) and active inflammatory bowel disease (IBD) on the stability of ACTB, B2M, GAPDH, HPRT1, SDHA, and TBP leukocyte expression. MATERIAL AND METHODS: Gene expression in controls and CRC and IBD patients (n = 21/18/25) was evaluated in real-time quantitative polymerase chain reaction (RT-qPCR) using NormFinder, geNorm, BestKeeper, and comparative ΔCt method, and validated by comparison with absolute quantification of interleukin 1ß (IL-1ß) and CCL4. RESULTS: HPRT1, SDHA and TBP were superior normalizers in CRC and IBD. The highest expression variability was noted in active IBD. B2M was significantly lower in CRC but higher in IBD. GAPDH was higher in CRC and IBD. ACTB and GAPDH corresponded with CRC advancement (ρ = 0.52 and ρ = 0.53) and with clinical activity in Crohn's disease (ρ = 0.44 and ρ = 0.57) and ulcerative colitis (GAPDH: ρ = 0.72). ACTB, B2M and GAPDH correlated with circulating inflammatory/angiogenic indices, differently in IBD and CRC. CONCLUSIONS: Leukocyte GAPDH, ACTB, and B2M expression is affected by bowel inflammation and cancer, rendering them unsuitable as a reference in CRC and IBD.