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.     

Variable expression of some "housekeeping" genes during human keratinocyte differentiation

We investigated the expression levels of four cellular "housekeeping" genes during epithelial differentiation. Differentiation is a dynamic process and various cellular RNAs have been targeted for use as internal controls during differentiation of human keratinocytes, but the consistent expression of such standards has not been previously validated. We used the organotypic (raft) culture system to grow stratified and differentiated epithelium in vitro. We compared cellular RNAs from epithelial tissues of both normal human keratinocytes and keratinocytes whose differentiation scheme is altered by the replication of human papillomavirus. Using ribonuclease protection assays to quantify RNA expression levels, we found that beta-actin and glyceraldehyde-3-phosphate dehydrogenase levels fluctuated during epithelial differentiation, whereas cyclophilin RNA and 28S-ribosomal RNA were the most consistently expressed during epithelial differentiation. These stably expressed cellular RNAs can be targeted as controls to permit quantitative expression analyses of cellular and pathogen RNAs during epithelial differentiation under various experimental conditions.     

(TG:CA)(n) repeats in human housekeeping genes

The unravelling of human genome sequence gives a new opportunity to investigate the role of repetitive sequences in gene regulation. Among the various types of repetitive sequences, the dinucleotide (TG:CA)(n) repeats are one of the most abundant in human genome and exhibit polymorphism. Early on, it was observed that the (TG:CA)(n) repeats could modulate gene expression and has the propensity to undergo conformational transitions in in vivo conditions. Recent reports describe the role of polymorphic (TG:CA)(n) repeats in gene regulation in several genes. In this work, we have analysed the distribution of (TG:CA)(n) (n >or= 6) repeats in human 'housekeeping genes' on which recently released Gene Chip data is available. Our results indicate that (i). The number of short intragenic (TG:CA)(n) repeats is significantly higher than the number of long repeats (ii). the proportion of genes with (TG:CA)(n) repeats (n >or= 12 units) had lower mean expression levels compared to those without these repeats, (iii). the genes belonging to the functional class of 'signalling and communication' had a positive association with repeats in contrast to the genes belonging to the 'information' class that were negatively associated with repeats.     

Mammalian housekeeping genes evolve more slowly than tissue-specific genes

Do housekeeping genes, which are turned on most of the time in almost every tissue, evolve more slowly than genes that are turned on only at specific developmental times or tissues? Recent large-scale gene expression studies enable us to have a better definition of housekeeping genes and to address the above question in detail. In this study, we examined 1581 human-mouse orthologous gene pairs for their patterns of sequence evolution, contrasting housekeeping genes with tissue-specific genes. Our results show that, in comparison to tissue-specific genes, housekeeping genes on average evolve more slowly and are under stronger selective constraints as reflected by significantly smaller values of Ka/Ks. Besides stronger purifying selection, we explored several other factors that can possibly slow down nonsynonymous rates in housekeeping genes. Although mutational bias might slightly slow the nonsynonymous rates in housekeeping genes, it is unlikely to be the major cause of the rate difference between the two types of genes. The codon usage pattern of housekeeping genes does not seem to differ from that of tissue-specific genes. Moreover, contrary to the old textbook concept, we found that approximately 74% of the housekeeping genes in our study belong to multigene families, not significantly different from that of the tissue-specific genes ( approximately 70%). Therefore, the stronger selective constraints on housekeeping genes are not due to a lower degree of genetic redundancy.     

Repetitive sequence environment distinguishes housekeeping genes

Housekeeping genes are expressed across a wide variety of tissues. Since repetitive sequences have been reported to influence the expression of individual genes, we employed a novel approach to determine whether housekeeping genes can be distinguished from tissue-specific genes by their repetitive sequence context. We show that Alu elements are more highly concentrated around housekeeping genes while various longer (> 400-bp) repetitive sequences (“repeats”), including Long Interspersed Nuclear Element-1 (LINE-1) elements, are excluded from these regions. We further show that isochore membership does not distinguish housekeeping genes from tissue-specific genes and that repetitive sequence environment distinguishes housekeeping genes from tissue-specific genes in every isochore. The distinct repetitive sequence environment, in combination with other previously published sequence properties of housekeeping genes, was used to develop a method of predicting housekeeping genes on the basis of DNA sequence alone. Using expression across tissue types as a measure of success, we demonstrate that repetitive sequence environment is by far the most important sequence feature identified to date for distinguishing housekeeping genes.     

Predicting housekeeping genes based on Fourier analysis

Housekeeping genes (HKGs) generally have fundamental functions in basic biochemical processes in organisms, and usually have relatively steady expression levels across various tissues. They play an important role in the normalization of microarray technology. Using Fourier analysis we transformed gene expression time-series from a Hela cell cycle gene expression dataset into Fourier spectra, and designed an effective computational method for discriminating between HKGs and non-HKGs using the support vector machine (SVM) supervised learning algorithm which can extract significant features of the spectra, providing a basis for identifying specific gene expression patterns. Using our method we identified 510 human HKGs, and then validated them by comparison with two independent sets of tissue expression profiles. Results showed that our predicted HKG set is more reliable than three previously identified sets of HKGs.     

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.     

高通量测序时代下持家基因定义的发展

持家基因一般指在所有细胞中均稳定表达的一类组成型基因,其产物是维持细胞基本生命活动所必需的。持家基因的定义及其发展,反映了其本质和时代特征。本文从基因表达水平量化技术的角度,结合作者利用转录组数据对猪持家基因的分析,讨论了持家基因的定义及其发展,尤其是技术的进步及数据量的剧增对持家基因的定义所引发的问题。本文总结发现,应该在研究技术的进步中,动态地认识持家基因定义所具有的层次性,从而对持家基因的定义不断赋予新的时代特征。     

Lateralization of housekeeping genes in the brain of one-day old chicks

Real-time quantitative PCR is an exceptionally sensitive method that can detect even very small differences in gene expression and, as such, it is essential to use suitable reference genes. Domestic chickens are used in a wide range of studies including neurobiology, behavior, ecology and disease transmission. In recent avian gene expression experiments, 18S (18S ribosomal RNA), beta actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have frequently been used; however, there is not enough evidence that these reference genes are suitable for all types of experiments. There is considerable evidence for lateralization in numerous learning tasks and for differences in the functional contribution of the two brain hemispheres. Therefore, the purpose of this study was to identify a set of reference genes for chick brain region called an intermediate medial mesopallium (IMM), which is connected with memory formation in the chick brain, whilst also taking into consideration the differences between the left and right hemispheres. This study evaluated the expression stability of eleven candidate housekeeping genes in the IMM region of the 1-day old chick brain. In our experimental system, the most reliable results were given by the NormFinder algorithm. The results show for the first time that ACTB, commonly used as an avian reference gene, is not suitable for investigation of gene expression in the chick brain and that brain lateralization exact selection of different reference gens for each hemisphere. For memory process investigations using tasks in one-day old chicks the most effective reference genes for the left hemisphere were HMBS and SDHA, and for the right hemisphere the most effective was RPL19.     

On the multimeric nature of natural human interleukin-6

Natural human interleukin-6 (IL-6) characterized under completely denaturing conditions consists of a set of differentially modified phosphoglycoproteins of molecular mass in the range from 23 to 30 kDa ("25-kDa" O-glycosylated species and "30-kDa" O- and N-glycosylated species). The 25-kDa O-glycosylated IL-6 (which contains only Ser- or Thr-GalNAc-Gal-NeuNAc and thus should not bind wheat germ or lentil lectins) bound to and was eluted from a wheat germ lectin affinity column by GlcNAc and from a lentil lectin affinity column by methyl-alpha-D-Man suggesting that the 25-kDa IL-6 species formed heteromeric complexes with the N-glycosylated 30-kDa IL-6. In non-denaturing gels (0.2% Nonidet P-40-polyacrylamide gel electrophoresis (PAGE)), even under reducing conditions (15 mM dithiothreitol or 1 M beta-mercaptoethanol and heating), fibroblast-derived IL-6 migrated as a predominant complex of mass approximately 85 kDa and additional minor 45-65-kDa complexes. Little IL-6 was detected in the size range 23-30 kDa. Elution of the major 85-kDa complex and re-electrophoresis through sodium dodecyl sulfate-PAGE revealed that it represented a heteromeric aggregate of the 25- and 30-kDa IL-6 species; the 45-65-kDa complexes were largely composed of the 25-kDa protein. The bulk of fibroblast-derived IL-6 eluted in the size range 45-85 kDa from a Sephadex G-200 gel filtration column further indicating that fibroblast-derived IL-6 was largely multimeric even in dilute solutions. Functionally, the high molecular mass IL-6 fractions from the G-200 column were less active in the B9 hybridoma growth factor assay than the lower molecular mass fractions but appeared to be equally active in the Hep3B hepatocyte-stimulating factor assay. Taken together, the data indicate that natural human IL-6 exists as a multimeric aggregate with varying biological activity.     

链霉菌看家基因引物的设计与验证

看家基因的扩增与测序是进行多基因系统进化分析首先需要解决的问题。针对链霉菌这一群高(G C)mol%革兰氏阳性细菌,选定4个看家基因:atpD、recA、rpoB和trpB,利用NCBI数据库中已有的2个链霉菌和3个分枝杆菌的全基因组序列,以及另两个链霉菌的recA基因序列,通过软件分析设计了各基因的扩增和测序引物,并优化了扩增反应条件。从所试验的55株链霉菌中,均特异地扩增出了上述4个基因的片段,并成功进行了序列测定,验证了所设计引物的实用性。所归纳的引物设计方法可用于高(G C)mol%革兰氏阳性细菌的其它看家基因,以促进多基因系统进化研究的开展。     

No chromosomal clustering of housekeeping genes in the marine chordate Ciona intestinalis

Housekeeping genes, widely expressed genes that are required for the basal function of most cell types, are clustered in the human and worm genomes. This arrangement suggests coordinate control of housekeeping gene expression at the chromosomal level. Here we examined whether this notion is applicable to a marine chordate, Ciona intestinalis. Using microarrays, we analyzed genes that were expressed in 11 organs of the adult, including the neural complex, branchial sac, esophagus, stomach, endostyle, intestine, body-wall muscle, heart, blood cells, ovary and testis. This analysis identified 158 genes that are expressed ubiquitously in these organs. These housekeeping genes could be classified into a range of Gene Ontology categories, in particular, ribosomal protein components. Of these 158 genes, we were able to map 141 genes onto the 14 pairs of the C. intestinalis chromosomes. They were distributed rather evenly over all the chromosomes, except for small clusters containing two or three genes. Therefore, the notion of chromosomal clustering of housekeeping genes is not applicable in this chordate.