Suitable Housekeeping Genes Research Articles

Molecular characterization and function of hif1a and fih1 in response to acute thermal stress in American shad (Alosa sapidissima).

In order to evaluate the function of hypoxia-inducible factor-1 alpha (hif1α) and factor inhibiting hif1α (fih1) in response to thermal stress, we first conducted a functional analysis of A. sapidissima hif1α and fih1, and determined hif1α and fih1 expressions in different tissues in response to thermal stress based on identified housekeeping genes (HKGs). The results showed that hif1α and fih1 were mainly located in the nucleus and cytoplasm. The full length cDNA sequence of hif1α and fih1 was 4073bp and 2759bp, respectively. The cDNA sequence of hif1α includes 15 exons encoding 750 amino acid residues, and the full length cDNA sequence of fih1 contains 9 exons encoding 354 amino acid residues. During the acute thermal stress transferring from 16 ± 0.5°C (control) to 20 ± 0.5°C, 25 ± 0.5°C, and 30 ± 0.5°C for 15min, it was found that the expression trends of hif1α and fih1 showed an inhibitory regulation in the heart, while they consistently expressed in brain, intestine, muscle, gill, kidney and liver. In conclusion, this is the first study to identify the tissue-specific HKGs in A. sapidissima and found that ef1α and β-actin are the most suitable HKGs. Hif1α and Fih1 are mainly the nuclear and cytoplasmic proteins, respectively, having high levels in the heart and brain. Alosa sapidissima countered a temperature increase from 16 to 25 ℃ by regulating the expressions of hif1α and fih1, but their physiological regulatory functions were unable to cope with acute thermal stress when the temperature difference was 14 ℃ (from 16 to 30 ℃).

Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System.

DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a "zero set" of potential reference gene candidates.

Using RNA-seq to identify suitable housekeeping genes for hypoxia studies in human adipose-derived stem cells

BackgroundHypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparativeDelta Ct, RefFinder, and the Livak method.ResultsGene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx and Hx conditions. From the dataset, 15 candidates with coefficient of variation ≤ 0.15 were identified, where differential expression analysis results further shortlisted 8 genes with least variation in expression levels. The top 4 housekeeping gene candidates, ALAS1, RRP1, GUSB, and POLR2B, were chosen for qRT-PCR validation. Additionally, 18S, a ribosomal RNA commonly used as housekeeping gene but not detected in the RNAseq method, was added to the list of housekeeping gene candidates to validate. From qRT-PCR results, 18S and RRP1 were determined to be stably expressed in cells cultured under hypoxic conditions.ConclusionsWe have demonstrated that 18S and RRP1 are suitable housekeeping genes for use in hypoxia studies with human adipose-derived stem cell and should be used in combination. Additionally, these data shown that the commonly used GAPDH and PGK1 are not suitable housekeeping genes for investigations into the effect of hypoxia in human adipose-derived stem cell.

Identification of suitable house-keeping genes during chikungunya virus infection.

Quantitative PCR (qPCR) is a reliable and robust technique for gene expression analysis, but its efficacy is dependent on the normalization of qPCR data with the stably expressed reference gene. Selection of a suitable reference gene is mandatory for accurate gene expression analysis, till data the most appropriate reference gene during chikungunya virus infection has not been elucidated. In this study the expression of reference genes(GAPDH, GUSB, HPRT, Beta-actin, 18S rRNA) was analysed during chikungunya virus infection by quantitative PCR. The stability of the house-keeping genes was evaluated with three bioinformatics softwares: BestKeeper, NormFinder and GeNorm. The significant variation in the expression of house-keeping genes (GusB, Beta-actin, HPRT) was observed during chikungunya virus infection; whereas GAPDH and 18S rRNA was most stable. The stability of reference genes analysed by the bioinformatics software further corroborate the results of qPCR. This is first study that identifies and validates the most suitable reference gene for normalization of qPCR data during chikungunya based gene expression analysis. This could serve as a reference study for the researchers working on different aspects of chikungunya virus infections.

Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.

Selection and validation of suitable reference gene for qPCR gene expression analysis in lamb testis cells under Sheep pox virus infection

Virus infection alters host gene expression, therefore ideal and stable reference housekeeping genes are required to normalise the expression of other expressed host genes in quantitative real-time PCR (qRT-PCR). The suitable reference gene may vary in response to different viral infections in different hosts or cells. In the present study, we cultured primary lamb testis cells (LTC) and assessed the expression stability of seven widely used housekeeping genes (B2M, HMBS, HPRT1, HSP-90, POLR2A, 18s_RNA, GAPDH) as reference genes in Sheeppox virus (SPPV) infected and control (uninfected-0h) LTC at 0.5h, 4.0h, 8.0h, and 12.0h post-infection) using NormFinder, Bestkeeper, geNorm, and the comparative ΔCT method in RefFinder based on their expression levels. Analysis revealed that HSP90, 18s_RNA, HPRT, POLR2A, and B2M were the most stable genes from the panel in the individual analysis group in 0h, 0.5h, 4.0h, 8.0h, and 12.0h, respectively. Furthermore, B2M was shown to be the most stable reference gene in the combined control with the respective and overall infected groups, except the control group of 4.0hpi of SPPV infection. In this study, we selected the most suitable reference genes in LTC for particular time points of SPPV infection. The identified most suitable housekeeping gene can be used during normalization of expression of other targeted genes at aspecific time point of SPPV infection.

Selection of Appropriate Housekeeping Genes for Gene Expression Normalization in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

Since lots of researches using qPCR technique have addressed gene expression in groupers, the suitable housekeeping genes are needed to obtain reliable and precise results. In this study, by using different mathematical algorithms (Genorm, normFinder, BestKeeper and comparative ΔCt method) and the comprehensive ranking software (RefFinder), the stability of seventeen housekeeping genes were assessed in eleven tissues of the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). Results showed that the stability of housekeeping genes were various in different tissues and the newly identified housekeeping genes were likely more stable than that of commonly used. In conclusion, the expression stability of housekeeping genes showed a tissue-specific manner, and the conserved oligomeric Golgi complex subunit 5 (cog5) and peroxisomal biogenesis factor 14 (pex14) could be universally used as appropriate housekeeping genes across different tissues in hybrid grouper. The present study provides the foundation for more accurate results of qPCR assay in hybrid grouper and other groupers.

Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene. The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases. It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.

Gene expression analysis for selection and validation of suitable housekeeping gene(s) in cadmium exposed pigeonpea plants inoculated with arbuscular mycorrhizae

The expression stability of six commonly used housekeeping genes (18S rRNA-18S ribosomal RNA, EF1α-elongation factor 1α, ACT1-Actin 1, GAPDH-Glyceraldehyde-3-phosphate dehydrogenase, TUB6-Tubulin/FtsZ family and UBC-Ubiquitin-conjugating enzyme) were scrutinized in leaves and roots of Cd stressed pigeonpea plants inoculated with arbuscular mycorrhizal (AM) species- Rhizoglomus intraradices (Ri), Funneliformis mosseae (Fm), Claroideoglomus etunicatum (Ce), C. claroideum (Cc). The stability profile of each gene was assessed using ΔCt, BestKeeper, NormFinder, RefFinder and geNorm algorithmic programs, which ranked different genes as most and least stable according to the tissues analysed. All the statistical algorithms ranked TUB6 as most stable and EF1α least stable housekeeping (HK) genes in both the plant tissues. The selected HK genes were verified using metallothionein (CcMT1) i.e. a stress responsive gene, whose expression altered under conditions of metal stress and AM inoculation. The expression pattern of CcMT1 varied highly when least stable reference gene was used for normalization as compared to most stable gene, under different treatments. Thus, there is a need of selecting suitable reference gene to achieve reliable results in gene expression studies using quantitative real time PCR (qRT-PCR). The study conducted will help future gene expression analysis in pigeonpea under specific stress.

FISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.

Identification of suitable mRNAs and microRNAs as reference genes for expression analyses in skin cells under sex hormone exposure.

Quantitative RT-PCR is the most accurate technique for the study of gene expression profiles, however, to ensure the accuracy of qPCR results, suitable reference genes are necessary for data normalization. Hormones influence the development and function of skin cells, regulating the expression of genes and miRNAs. Nevertheless, the stability of reference genes after sex hormone treatment has not been thoroughly investigated. In this study, we evaluated the expression of a set of candidate mRNAs and microRNsA (miRNA) as reference genes in keratinocytes (HaCaT cells), primary human fibroblasts and a melanoma cell line (LM-36 cells) under testosterone or 17β-estradiol treatment. Two algorithms, namely geNorm, Best-Keeper, and the comparative ΔCt method were used to evaluate the expression stability of the candidate reference genes. The comprehensive ranking showed that TBP and miR-191-5p are the most stable expressed genes across all cultured cells under hormone treatment. Furthermore, we observed that GAPDH, HPRT1 and U6 snRNA expression may be altered by hormone exposure, thus, these genes are not recommended as reference genes. In conclusion, the present study provides, to the best of our knowledge, the first evaluation of expressed mRNA(s) and miRNA(s) as reference genes in three different types of skin cells under the stimulation of sex hormones.

Molecular Detection and Quantification Gene Expression of Efflux Pump Antibiotic Resistance Genes in Extensive Drug Resistance Pseudomonas Aeruginosa Isolated from Clinical Infection Patients in Al Diwaniyah City of Iraq

The study was included investigated 16 extensive drug resistance pseudomonas aeruginosa isolateswhich previously isolated from sputum, burns, urine, and ear swab clinical patients samples. The reversetranscription Real Time PCR (RT-qPCR) was performed for molecular detection and quantification of geneexpression levels for efflux pump antibiotic resistance gene (mexA, mexB, mexC, mexE, and mexY) andchromosomal lactamase (AmpC) gene, as well as outer membrane prion protein (OprD) gene in extensivedrug pseudomonas aeruginosa isolates. The relative expression analysis was done by used the 2-??CT (Livak)method, this method was used for normalization of expression levels (mRNA transcript levels) in targetgenes with suitable housekeeping gene. In this study 30S ribosomal protein S12 (rpsL) housekeeping genewas used in normalization of antibiotic resistance genes expression levels in extensive drug pseudomonasaeruginosa isolates. The efflux pump resistance gene expression were by RT-qPCR technique. The relativegene expression analysis were showed highest frequency of overexpression at (100%, 100%, 87.5%,81.25%, 56.25%) for (mexB, mexE, mexA,mexC, and mexY) with fold change ranged (2.16- 184.82) foldchange compared to wild type PA control isolate. The relative gene expression analysis of chromosomallactamase AmpC gene was showed highest frequency of overexpression (100%) in XDR isolates withexpression levels ranged (6.77- 73.1) fold change compared to wild type PA control isolate. Whereas, therelative gene expression analysis of OprD gene was showed (68.75%) frequency of down expression inXDR Pseudomonas aeruginosa isolates with expression levels ranged (0.01-0.16) fold change compared towild type PA control isolate. In conclusion, The XDR Pseudomonas aeruginosa isolates can ability to overexpression production of inducible chromosomal lactamase AmpC gene, efflux pumps protein genes, andlow expression outer membrane prion protein (OprD) gene as important antimicrobial mechanism againstCephalosporins and Carbapenems antibiotic classes.

Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries.

Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. RNA was isolated from 5 normal human ovaries (52-79years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49-75years of age), and 4 PCOS ovaries (18-35years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.

Selection and validation of suitable reference genes for quantitative real time PCR analysis of gene expression studies in patchouli under Meloidogyne incognita attack and PGPR treatment

Patchouli (Pogostemon cablin) is an economically important aromatic plant and is often vulnerable to nematode attack (Meloidogyne incognita). The application of PGPR has tremendously changing disease management approach in plants and a systematic understanding the molecular mechanism behind it is needed to control over the disease more precisely. Since, qRT-PCR is the most frequently used molecular tools in studying molecular mechanism but is greatly dependent upon suitable reference or housekeeping genes. This study is aim to select suitable reference genes for qRT-PCR experiments in patchouli under the condition of nematode attack and PGPR treatments. Expression pattern of six reference genes (Actin, EF1, ACP, COX3, GAPDH and Pat18S) were statistically analyzed and validated using different algorithms viz., ΔCt, BestKeeper, GeNorm, NormFinder and RefFinder. The result showed that the Actin and ACP are the most suitable reference genes and the combination of ACP &GAPDH gene pairs can also be used whenever the experiment needed.

Identification of housekeeping genes for microRNA expression analysis in kidney tissues of Pkd1 deficient mouse models

Polycystic kidney disease is a complex clinical entity which comprises a group of genetic diseases that leads to renal cyst development. We evaluated the most suitable housekeeping genes for microRNA expression by RT-qPCR analyses of kidney tissues in Pkd1-deficient mouse models from a panel of five candidates genes (miR-20a, miR-25, miR-26a, miR-191 and U6) and 3 target genes (miR-17, miR-21 and let-7a) using samples from kidneys of cystic mice (Pkd1flox/flox:Nestincre, CY), non-cystic controls (Pkd1flox/flox, NC), Pkd1-haploinsufficient (Pkd1+/−, HT), wild-type controls (Pkd1+/+, WT), severely cystic mice (Pkd1V/V, SC), wild-type controls (CO). The stability of the candidate genes was investigated using NormFinder, GeNorm, BestKeeper, DataAssist, and RefFinder software packages and the comparative ΔCt method. The analyses identified miR-26a as the most stable housekeeping gene for all kidney samples, miR-20a for CY and NC, miR-20a and miR-26a for HT and WT, and miR-25 and miR-26a for SC and CO. Expression of miR-21 was upregulated in SC compared to CO and trends of miR-21 upregulation and let-7a downregulation in CY and HT compared to its control kidneys, when normalized by different combinations of miR-20a, miR-25 and miR-26a. Our findings established miR-20a, miR-25, and miR-26a as the best housekeeping genes for miRNA expression analyses by RT-qPCR in kidney tissues of Pkd1-deficient mouse models.

Transcriptome-based evaluation and validation of suitable housekeeping gene for quantification real-time PCR under specific experiment condition in teleost fishes

Quantification real-time PCR (qRT-PCR) is a common method in analysis of gene expression, but the stable reference genes for the normalization analysis have not been appreciated before identifying expression pattern of genes in teleost fishes. In this study, we selected eight candidate reference genes (18S, Actin, EF-1α, 40S, B2M, TUBA, UBCE and GAPDH) basing on transcriptome analysis and the traditional housekeeping genes, and analyzed the stability of the reference genes in spleen, head kidney and head kidney leukocytes (HKL) after pathogen challenge in Schizothorax prenanti (S. prenanti). Three common programs (geNorm, NormFinder and Bestkeeper) were used to evaluate the stability of the candidate reference genes. Two reference genes, Actin and EF-1α presented higher stability, while 18S and GAPDH were the lower stable genes, both in in vitro and in vivo. An important immune gene, toll-like receptor 22a (TLR22a), was selected to validate the stability of the proposed reference genes (Actin and EF-1α) across different experiment treatments. The results reveal that Actin and EF-1α are quite suitable reference genes for the normalization analysis. Otherwise, using the most stable gene Actin to validate the reliable of transcriptome data showed the high correlation between the fold change of transcriptome data and qRT-PCR data. In conclusion, our study not only acquired the suitable reference gene for the qRT-PCR assay under specific experiment condition, but also provided a comprehensive method to evaluate and validate the reference gene based on transcriptome analysis in teleost fishes.

Validation of suitable housekeeping genes for 3T3-L1 derived adipocytes cultured in obesity mimicking conditions and RAW 264.7 macrophage cells lines in hypoxic and normoxic conditions

Abstract Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) is the most commonly used method for gene expression analysis, and reliable results depend on proper normalization by stable reference genes. Adipose tissue hypoxia is the new concept in understanding the major disorders associated with obesity and may facilitate cellular mechanisms associated with obesity like chronic inflammation, macrophage infiltration, adiponectin reduction, leptinelevation, adipocyte death, ER stress and mitochondrial dysfunction. The hypoxic conditions in the obese adipose tissue lead to differential expression of genes in the resident cells of adipose tissues including macrophages andhence reliable normalization of RT-qPCR data becomes crucial. In this study, the expression stability of 4 most commonly used candidate reference genes, ACTB, B2M, HMBS and 18SrRNA were evaluated in murine adipocytesand macrophage cell lines, in presence of high free fatty acids, under hypoxia and normoxia conditions. The results were analyzed by manually calculating fold difference using 2−ΔΔCt and validated by using two algorithms, NormFinder, and Bestkeeper. Our results showed HMBS and B2M as the most stably expressed genes in both cell lines under obesity mimicking conditions (in presence of high amount of FFAs and hypoxia). By contrast, ACTB and 18S rRNA were the least stable genes under the tested conditions. Furthermore, the expression levels of FABP4 andPPAR-γ (for adipocytes) and MCP-1 and IL-6 (for macrophages) were profiled in these cells to validate the selected reference genes. Our data provides a basis for gene expression analysis in future studies related to inflammation in hypoxia related obesity pathogenesis.

Identification of housekeeping genes as references for quantitative real-time RT-PCR analysis in Misgurnus anguillicaudatus

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a well-known method to quantify gene expression by comparing with the reference genes. Generally, housekeeping genes were set as references, as for their stable expression in varying conditions. Here, we try to evaluate few of such genes to identify suitable housekeeping genes as references for qRT-PCR analysis of gene expression in Misgurnus anguillicaudatus. This study evaluated the expression of four commonly used housekeeping genes, i.e. b-actin (ACTB), elongation factor 1 alpha (EF-1a), glyceraldehyde-3-phosphate (GAPDH) and 18S ribosomal RNA (18S rRNA), in gender difference, effects of tissue type, different developmental stages, chemical treatment of embryos/larvae with commonly used vehicles for administration and agents that represent known environmental toxicant. Rank ordering of expression stability was done using geNorm, NormFinder and BestKeeper algorithms. Results suggested that in the qRTPCR test, in all the experimental conditions, EF-1a could be selected as reference gene when analysing a target gene. For the study of different development stages, ACTB could be a candidate as reference gene. For the studies associated with different gender and tissue types, EF-1a would be better targeted as reference gene. Meanwhile, in toxicant treatment, expression of EF-1a seems to be more stable than others and could be considered as reference gene. This study could provide useful guidelines that can be expected to aid M. anguillicaudatus researchers in their initial choice of housekeeping genes for future studies and enable more accurate normalization of gene expression data.

Rapid Communication: MiR-92a as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

Our aim was to identify a suitable microRNA housekeeping gene for real-time PCR analysis of bovine mastitis-related microRNA in milk. We identified , , and as housekeeping gene candidates on the basis of previous Solexa sequencing results. Threshold cycle (CT) values for , , and did not differ between milk from control cows and milk from mastitis-affected cows. NormFinder software identified as the most stable single housekeeping gene. We evaluated the suitability of the housekeeping gene candidates by using them to assess expression levels of the inflammation-related gene . Regardless of the housekeeping gene candidates used for normalization, relative expression levels of were significantly higher in mastitis-affected samples than in control samples. However, of all the housekeeping genes and gene combinations investigated, normalization with alone generated the difference in relative expression between mastitis-affected and control samples with the highest significance. These results suggest that is suitable for use as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity

Oncogenic microRNAs (miRs) have emerged as diagnostic biomarkers and novel molecular targets for anti-cancer drug therapies. Real-time quantitative PCR (qPCR) is one of the most powerful techniques for analyzing miRs; however, the use of unsuitable normalizers might bias the results. Tumour heterogeneity makes even more difficult the selection of an adequate endogenous normalizer control. Here, we have evaluated five potential referenced small RNAs (U6, rRNA5s, SNORD44, SNORD24 and hsa-miR-24c-3p) using RedFinder algorisms to perform a stability expression analysis in i) normal colon cells, ii) colon and breast cancer cell lines and iii) cancer stem-like cell subpopulations. We identified SNORD44 as a suitable housekeeping gene for qPCR analysis comparing normal and cancer cells. However, this small nucleolar RNA was not a useful normalizer for cancer stem-like cell subpopulations versus subpopulations without stemness properties. In addition, we show for the first time that hsa-miR-24c-3p is the most stable normalizer for comparing these two subpopulations. Also, we have identified by bioinformatic and qPCR analysis, different miR expression patterns in colon cancer versus non tumour cells using the previously selected suitable normalizers. Our results emphasize the importance of select suitable normalizers to ensure the robustness and reliability of qPCR data for analyzing miR expression.