Validation of suitable internal control genes for expression studies in aging.

Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors

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

IDENTIFIKASI GEN HOUSEKEEPINGPADA DNA GENOM IKAN GURAME (Osphronemusgouramy)

Gen housekeeping merupakan gen pengatur fungsi selular basal pada organisme hidup yang secara konstitutif diekspresikan terus menerus dalam kondisi apapun. Oleh karena sifat tersebut, gen housekeepingdigunakansebagaikontrol internalpadaanalisisekspresi gen lain,dan merupakan instrumental untukkalibrasi di banyakaplikasibioteknologidanpenelitiangenom. Namun informasi genetik mengenai gen housekeeping pada ikan gurame (Osphronemus gouramy) sangat minim, sehingga penelitian ini bermaksud untuk mengisolasi dan mengidentifikasi gen-gen housekeeping pada ikan gurame dengan merancang primer degenerate yang dapat mengamplifikasi gen housekeeping pada DNA genom ikan gurame. Primer degenerate dirancang berdasarkan konsensus sikuen gen dari beberapa ikan Infraclassis Teleostei. Terlebih dahulu kandidat gen housekeeping dipilih berdasarkan studi literatur, dan diperoleh tujuh kandidat gen 18S rRNA, ODC1, HPRT1, TBP, UBC, G6PD, dan TAF2. Perancangan primer dilakukan secara online pada laman Primaclade. Amplifikasi gen housekeeping pada ikan gurame dilakukan menggunakan metode nested PCR yang mana terdapat dua set pasangan primer terdiri dari outer-primer dan primer nested. Dua primer yang mampu mengamplifikasi gen housekeeping pada DNA genom ikan gurame dilakukan sequencing untuk mengetahui urutan sikuen gen. Hasil identifikasi gen 18S rRNA dan ODC1 dari ikan gurame (Osphronemus gouramy) berdasarkan analisis BLAST dan pohon filogenetik menunjukkan semua gen yang diperoleh sesuai dengan gen target yang diharapkan dan mampu diekspresikan pada ikan gurame. Analisis BLAST pada pasangan primer spesifik yang dirancang menunjukkan gen 18S rRNA dan ODC1 spesifik pada ikan gurame;---Housekeeping genes are regulating basal cellular function genes in living organisms that are assumed constitutively to be expressed continuously under any conditions. Because of these properties, housekeeping genes are used as internal controls in the analysis of other gene expressions, and are instrumental for calibration in many applications of biotechnology and genome research. Inadequacy of genetic information about housekeeping genes in goramy (Osphronemus gouramy), a study was conducted to isolate and identification housekeeping genes in goramy by designing degenerates primer pair that can amplify housekeeping genes in the goramy genome. The degenerate primer pair was designed based on the consensus of the gene sequences of several Teleostei fish. Firstly, housekeeping gene candidates were selected based on literature studies, and seven candidates of genes 18S rRNA, ODC1, HPRT1, TBP, UBC, G6PD, and TAF2 were obtained. The primer design has been done online on the Primaclade page. Amplification of housekeeping genes in goramywere using the nested PCR method which has two sets of primer pairs consisting of outer-primers and nested primers. Only two primers capable of amplifying housekeeping genes in goramy genom. The results of 18S rRNA and ODC1 genes identification from goramy (Osphronemus gouramy) based on BLAST analysis and phylogenetic trees showed that all genes obtained were in accordance with the expected target genes and were able to be expressed in goramy. BLAST analysis on the specific primer pairs shows that the 18S rRNA and ODC1 genes specific to goramy

Do Housekeeping Genes Exist?

The searching of human housekeeping (HK) genes has been a long quest since the emergence of transcriptomics, and is instrumental for us to understand the structure of genome and the fundamentals of biological processes. The resolved genes are frequently used in evolution studies and as normalization standards in quantitative gene-expression analysis. Within the past 20 years, more than a dozen HK-gene studies have been conducted, yet none of them sampled human tissues completely. We believe an integration of these results will help remove false positive genes owing to the inadequate sampling. Surprisingly, we only find one common gene across 15 examined HK-gene datasets comprising 187 different tissue and cell types. Our subsequent analyses suggest that it might not be appropriate to rigidly define HK genes as expressed in all tissue types that have diverse developmental, physiological, and pathological states. It might be beneficial to use more robustly identified HK functions for filtering criteria, in which the representing genes can be a subset of genome. These genes are not necessarily the same, and perhaps need not to be the same, everywhere in our body

A comprehensive functional analysis of tissue specificity of human gene expression

<p>Abstract</p> <p>Background</p> <p>In recent years, the maturation of microarray technology has allowed the genome-wide analysis of gene expression patterns to identify tissue-specific and ubiquitously expressed ('housekeeping') genes. We have performed a functional and topological analysis of housekeeping and tissue-specific networks to identify universally necessary biological processes, and those unique to or characteristic of particular tissues.</p> <p>Results</p> <p>We measured whole genome expression in 31 human tissues, identifying 2374 housekeeping genes expressed in all tissues, and genes uniquely expressed in each tissue. Comprehensive functional analysis showed that the housekeeping set is substantially larger than previously thought, and is enriched with vital processes such as oxidative phosphorylation, ubiquitin-dependent proteolysis, translation and energy metabolism. Network topology of the housekeeping network was characterized by higher connectivity and shorter paths between the proteins than the global network. Ontology enrichment scoring and network topology of tissue-specific genes were consistent with each tissue's function and expression patterns clustered together in accordance with tissue origin. Tissue-specific genes were twice as likely as housekeeping genes to be drug targets, allowing the identification of tissue 'signature networks' that will facilitate the discovery of new therapeutic targets and biomarkers of tissue-targeted diseases.</p> <p>Conclusion</p> <p>A comprehensive functional analysis of housekeeping and tissue-specific genes showed that the biological function of housekeeping and tissue-specific genes was consistent with tissue origin. Network analysis revealed that tissue-specific networks have distinct network properties related to each tissue's function. Tissue 'signature networks' promise to be a rich source of targets and biomarkers for disease treatment and diagnosis.</p

Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

BACKGROUND: Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. RESULTS: Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). CONCLUSION: Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided

Housekeeping gene validation for RT-qPCR studies on synovial fibroblasts derived from healthy and osteoarthritic patients with focus on mechanical loading

Selection of appropriate housekeeping genes is essential for the validity of data normalization in reverse transcription quantitative PCR (RT-qPCR). Synovial fibroblasts (SF) play a mediating role in the development and progression of osteoarthritis (OA) pathogenesis, but there is no information on reliable housekeeping genes available. Therefore the goal of this study was to identify a set of reliable housekeeping genes suitable for studies of mechanical loading on SF from healthy and OA patients. Nine genes were evaluated towards expression stability and ranked according their relative stability determined by four different mathematical procedures (geNorm, NormFinder, BestKeeper and comparative ΔCq). We observed that RPLP0 (ribosomal protein, large, P0) and EEF1A1 (eukaryotic translation elongation factor 1 alpha 1) turned out to be the genes with the most stable expression in SF from non-OA or OA patients treated with or without mechanical loading. According to geNorm two genes are sufficient for normalization throughout. Expression of one tested target gene varied considerably, if normalized to different candidate housekeeping genes. Our study provides a tool for accurate and valid housekeeping gene selection in gene expression experiments on SF from healthy and OA patients with and without mechanical loading in consistent with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and additionally demonstrates the impact of proper housekeeping gene selection on the expression of the gene of interest

Differential Selective Constraints Shaping Codon Usage Pattern of Housekeeping and Tissue-specific Homologous Genes of Rice and Arabidopsis

Intra-genomic variation between housekeeping and tissue-specific genes has always been a study of interest in higher eukaryotes. To-date, however, no such investigation has been done in plants. Availability of whole genome expression data for both rice and Arabidopsis has made it possible to examine the evolutionary forces in shaping codon usage pattern in both housekeeping and tissue-specific genes in plants. In the present work, we have taken 4065 rice–Arabidopsis homologous gene pairs to study evolutionary forces responsible for codon usage divergence between housekeeping and tissue-specific genes. In both rice and Arabidopsis, it is mutational bias that regulates error minimization in highly expressed genes of both housekeeping and tissue-specific genes. Our results show that, in comparison to tissue-specific genes, housekeeping genes are under strong selective constraint in plants. However, in tissue-specific genes, lowly expressed genes are under stronger selective constraint compared with highly expressed genes. We demonstrated that constraint acting on mRNA secondary structure is responsible for modulating codon usage variations in rice tissue-specific genes. Thus, different evolutionary forces must underline the evolution of synonymous codon usage of highly expressed genes of housekeeping and tissue-specific genes in rice and Arabidopsis