Suitable Housekeeping Genes Research Articles

Effects of pesticide compounds (chlorothalonil and mancozeb) and benzo[a]pyrene mixture on aryl hydrocarbon receptor, p53 and ubiquitin gene expression levels in haemocytes of soft-shell clams (Mya arenaria)

The aim of this study is to investigate the effects of the pesticides/polycyclic aromatic hydrocarbon mixture on aryl hydrocarbon receptor (AhR), p53 and ubiquitin mRNA level in haemocytes of Mya arenaria exposed to a mixture of chlorothalonil, mancozeb and benzo[a]pyrene (BaP) for 48 and 72 h. AhR, p53 and ubiquitin gene expression levels were quantified using quantitative Real-time PCR. For robust and accurate quantification of transcripts, suitable housekeeping genes were selected from four sets of ribosomal and elongation factors transcripts previously sequenced from Mya arenaria using geNorm open source software. Quantitative Real-time PCR data exhibited a significantly high expression of AhR after 72 h of exposure (P ≤ 0.05). p53 gene expression seems to be up-regulated by the mixture after 48 h, however not significantly; but the level of p53 mRNA is down-regulated by the xenobiotics between 48 and 72 h after exposure. This study postulates that AhR mRNA levels could be used as an indicator of the exposure of clams' haemocytes to a mixture of xenobiotics such as chlorothalonil, mancozeb and BaP. However, further studies have to be pursued in order to unravel the molecular mechanisms involved in the p53 signaling pathway.

Molecular Markers of Injury in Kidney Biopsy Specimens of Patients with Lupus Nephritis

Prediction of prognosis in patients who have lupus nephritis is inadequate, limiting individualization of potentially toxic therapy. Advances in tissue molecular techniques offer new approaches to study mechanisms underlying kidney injury, and add to prognostic information gleaned from biopsy specimens. Analysis of mRNA expression in formalin-fixed, paraffin-embedded renal biopsy specimens is limited by both quantity and quality of RNA, requiring RNA pre-amplification, which can introduce bias. Accordingly, we developed a new technique for RNA extraction from human kidney formalin fixed paraffin embedded biopsy specimens, and used Taqman low-density arrays Applied Biosystems, Carlsbad, CA to simultaneously measure 48 mRNAs in duplicate, in a single biopsy. We extracted mRNA from more than 150 blocks to determine the quantity and vintage of biopsy tissue suitable for analysis using this protocol. We then used Taqman low-density arrays to identify suitable housekeeping genes in lupus nephritis. Finally, we measured expression of 48 mRNA transcripts in archived lupus biopsy specimens (n = 54). We identified that the mRNA levels of three transcripts (MMP7, EGF, COL1A1) relate to pathological indices of kidney injury and kidney function at the time of biopsy; these were associated with parallel changes in expression of these proteins. This new method for measurement of kidney biopsy mRNA expression has enabled us to identify tissue biomarkers of kidney damage and function, and potentially can increase the information yielded from diagnostic kidney biopsy specimens to improve tailoring of therapy.

Validation of housekeeping genes as internal controls for studying biomarkers of endocrine-disrupting chemicals in disk abalone by real-time PCR

Our experiments were designed to identify suitable housekeeping genes (HKGs) in disk abalone as internal controls to quantify biomarker expression following endocrine disrupting chemicals (EDCs). Relative expression levels of twelve candidate HKGs were examined by real-time reverse transcription PCR (qRT-PCR) in gill and hepatopancreas of abalone following a 7-day challenge with either tributyltin chloride (TBT) or 17β-estradiol (E2). The expression levels of several conventional HKGs, such as 18s rRNA, glyceraldehyde-3-phosphate dehydrogenase and β-actin, were significantly altered by the challenges, indicating that they might not be suitable internal controls. Instead, the geNorm analysis pinpointed ribosomal protein L-5/ elongation factor 1 and ribosomal protein L-5/ succinate dehydrogenase as the most stable HKGs under TBT and E2 challenges, respectively. Moreover, these three HKGs also showed the highest stabilities overall amongst different tissues, genders and EDC challenges. The expression of a biomarker gene, cytochrome P450 4B (CYP4), was also investigated and exhibited a significant increase after the challenges. Importantly, when unsuitable HKGs were used for normalization, the influence of two EDCs on CYP4 expression was imprecisely overestimated or underestimated, which strongly emphasized the importance of selecting appropriately validated HKGs as internal controls in biomarker studies.

Validation of reference genes for real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang

Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.

Identification of genes from flat oyster Ostrea edulis as suitable housekeeping genes for quantitative real time PCR

Bonamia ostreae is an intrahaemocytic protozoan affecting Ostrea edulis. The parasite multiplies within haemocytes without being degraded and involves changes in cellular activities. Studies aiming at better understanding host response to a pathogen at the transcriptome levels are frequently based on the use of real time PCR assays, which require some reference genes. However, very few sequence data is available for O. edulis in public databases. Subtracted cDNA libraries were constructed from the O. edulis haemocytes in order to identify genes involved in host reactions against the parasite and quantitative real time PCR assays were developed to study expression of these genes. In this context, identification of reference genes and study of their relative expression stability were required for quantitative real time PCR normalization. The expression of 5 potential candidate reference genes from O. edulis (ie elongation factor 1 alpha (EF1-α), 60S ribosomal protein L5 (L5), glyceraldehyde 3-phosphate-dehydrogenase (GAPDH), polyubiquitin (Ubiq) and β-actin (ACT)) was studied using RNAs extracted from pools of haemocytes in contact with the parasite B. ostreae and haemocytes alone. Gene expression was quantified by real time PCR and expression stability was analysed with two analytical approaches GeNorm and NormFinder. GAPDH and EF1-α were identified as the most stable genes with the GeNorm analysis. Whatever were the tested conditions, EF1-α was also found as the most stable gene using Normfinder. The less stable gene was β-actin although this gene is commonly used as housekeeping gene in many studies. Our results suggest using GAPDH and EF1-α combined as reference genes when studying expression levels in haemocytes of O. edulis. In addition, the complete ORF of these two genes was characterized.

ABCB1 gene expression in peripheral blood mononuclear cells in healthy Thai males and females

We compared the expression of the ABCB1 gene in healthy male and female Thai subjects; this gene encodes the P-glycoprotein transporter in peripheral blood mononuclear cells (PBMCs). We also identified the most suitable housekeeping genes for normalization of ABCB1 expression levels in PBMCs. PBMCs from 30 females and 26 males were isolated. Total RNA was extracted, followed by reverse transcription (100 ng total RNA per sample). The internal normalization controls were actin-beta, beta-2M and GAPDH. Real-time quantitative PCR was then performed to determine the expression levels of the ABCB1 gene. The expression levels were found to be 1.5- to 2.5-fold higher in males, depending on the endogenous control used for normalization. Actin-beta was the most stable control gene and could be used as a single endogenous control for normalization of ABCB1 expression levels in PBMCs. However, more than one endogenous control genes are recommended for normalization of gene expression. We conclude that the expression levels of ABCB1 in PBMCs is influenced by gender; this helps, in part, explain the gender difference in pharmacokinetics and pharmacodynamics of drugs that are P-glycoprotein substrates. ABCB1 gene expression profiles need to be carefully interpreted with regards to the endogenous control genes that are involved.

An RNA spiking method demonstrates that 18S rRNA is regulated by progesterone in the mouse uterus

Identifying suitable housekeeping genes for quantitative RT-PCR in the uterus is problematic, as this tissue undergoes significant structural and functional alterations during the oestrous cycle and pregnancy in response to circulating hormones. The suitability of 18S rRNA as a housekeeping gene in mouse uterus was investigated by introducing an 'RNA spike' standard into the reverse transcription reaction. 18S rRNA levels increased by Day 4 of pregnancy and after progesterone administration in ovariectomized mice. We conclude that 18S rRNA is not a suitable housekeeping gene for quantitative RT-PCR analysis in progesterone-responsive tissues, and the RNA spiking method provides a suitable alternative.

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

BackgroundConsidering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.ResultsHere we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold.ConclusionAltogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation

Real-time RT-PCR is currently the most sensitive, specific and precise approach to analyse gene expression changes in plant stress studies. The determination of biologically meaningful transcript quantities requires accurate normalisation of the raw data. During relative quantification the reliability of the results depends on the stable expression of the endogenous control genes across the experimental samples. Four widely used internal control genes (cyclophilin, elongation factor 1α, polyubiquitin, tubulin β-chain) and two potential candidates (serine/threonine-protein phosphatase 2A and ubiquitin-conjugating enzyme) genes were assessed under Cd-stress and at different developmental stages in leaves of Populus jacquemontiana D. var. glauca H. Complementary DNA (RiboGreen) based quantification method revealed variations in the expression level of reference genes. The variability was more pronounced under severe stress conditions. Less variation was observed in the case of ef-1α, pp2a and ubc10. Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the RiboGreen method or by using multiple references. The impact of variability of reference gene on the target gene evaluation was demonstrated. It was proved that in the absence of suitable housekeeping genes, for example under severe stress, RiboGreen method is convenient tool for transcript normalisation.

Selection of housekeeping genes for gene expression studies on the development of fruit bearing shoots in Chinese jujube (Ziziphus jujube Mill.)

Chinese jujube (Ziziphus jujube Mill.) is important economically for its fruit and also bears attractive medical value. Its flower development concurs with the growth of current fruit bearing shoots (FBSs). However, events involved in current FBSs, including FBS differentiation, flower development, and reliable housekeeping gene are still unknown. In this study, the morphology of FBS development and floral formation were examined and expression profiles of several potential internal control genes was determined. These included genes encoding proteins involved in protein translation (translation elongation factor 1alpha, ZjEF1), protein folding (cyclophilin, ZjCyP) or degradation (ubiquitin extension protein, ZjUBQ), and proteins involved in the structure of the cytoskeleton (beta-actin, ZjACT) or nucleosome (histone3, ZjH3). Our results showed that the floral development in early growing FBSs (less than 20 mm in length) or shoot apices was not complete. Among ZjACT, ZjEF1, ZjCyP, ZjUBQ, and ZjH3, ZjH3 was the most suitable housekeeping gene to evaluate FBS development, based on their expression in early growing FBSs, shoot apices, and different organs. These results will be useful for further molecular mechanism study about FBS development in Chinese jujube.

Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells

The stability of housekeeping genes (HKGs) is critical when performing real-time quantitative PCR. To date, the stability of common HKGs has not been systematically compared in human airway epithelial cells (AEC) in normal and atopic subjects. Expression levels of 12 HKGs were measured in AECs from a cohort of 30 healthy atopic nonasthmatic or atopic asthmatic children. Gene expression stability was determined using three different Visual Basic for Applications applets (geNorm, NormFinder and BestKeeper). All 12 HKGs were expressed in AECs. However, the hypoxanthine ribosyltransferase and TATA-binding protein genes were excluded from further analysis due to low expression levels. The cyclophilin A gene was ranked the most stable by all three methods. The expression levels of the beta-actin and glyceraldehyde-3-phosphate dehydrogenase genes were significantly different between the three groups of patients, with atopic asthmatics showing the highest expression levels for both genes. The results suggest that the cyclophilin A gene is the most suitable housekeeping gene analysed for expression studies utilising uncultured bronchial airway epithelial cells from healthy and asthmatic children, and highlight the importance of validating housekeeping genes for each experimental model.

Selection of reference genes for real-time PCR in human hepatocellular carcinoma tissues

To investigate the most suitable housekeeping genes for quantifying a change in mRNA expression levels due to hepatitis virus B related hepatocellular carcinoma (HCC). Expression of mRNA encoding ACTB, GAPDH, B2M, HPRT and TBP was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in matched malignant and non-malignant tissues obtained from 65 non-treated HCCs. The software programs geNorm and NormFinder were used to ascertain the most suitable reference gene combination. All candidate genes showed significantly different expression between malignant and non-malignant samples. GAPDH and ACTB, genes most frequently used for normalization, were heavily regulated during HCC carcinogenesis and progression. B2M expression levels varied with hepatitis infection status. The combination of HPRT and TBP expression levels were the most stable, regardless of differences in tumor stage and cirrhotic and malignancy status. It is necessary to select reference genes based on tissue and disease specific expression profile and to further identify novel reference genes with greater expression stability for use in HBV related HCC gene expression studies.

Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

BackgroundDue to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan® PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).ResultsT-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 ± 0.33, without significant differences between self-designed and ABI inventoried Taqman® gene assays. Only two of the tested Taqman® ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.ConclusionIn comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Determination of suitable housekeeping genes for normalisation of quantitative real time PCR analysis of cells infected with human immunodeficiency virus and herpes viruses

The choice of an appropriate housekeeping gene for normalisation purposes has now become an essential requirement when designing QPCR experiments. This is of particular importance when using QPCR to measure viral and cellular gene transcription levels in the context of viral infections as viruses can significantly interfere with host cell pathways, the components of which traditional housekeeping genes often encode. In this study we have determined the reliability of 10 housekeeping genes in context of four heavily studied viral infections; human immunodeficiency virus type 1, herpes simplex virus type 1, cytomegalovirus and varicella zoster virus infections using a variety of cell types and virus strains. This provides researchers of these viruses with a shortlist of potential housekeeping genes to use as normalisers for QPCR experiments.

Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray

BackgroundDuring systemic gram-negative bacterial infections, lipopolysaccharide (LPS) ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg) using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR).ResultsTwelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg) and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep.ConclusionWe have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.

Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference

A novel two-step real-time RT-PCR assay using SYBR ® Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5′-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.

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

BackgroundControl 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.ResultsInitial 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).ConclusionUsing 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.

Gene expression of single articular chondrocytes

Although previous studies in the field of tissue engineering have provided important information about articular cartilage, their conclusions are based on population averages and do not account for variations in cell subpopulations. To obtain a precise understanding of chondrocytes, we investigated the effects of cartilage zone and seeding duration on single chondrocyte gene expression to select an optimal zone for tissue engineering (Phase I), followed by an evaluation of growth factor exposure on the zone selected in Phase I (Phase II). In Phase I, superficial and middle/deep bovine articular chondrocytes were seeded in monolayers for 3 or 18 h. In Phase II, middle/deep chondrocytes (selected in Phase I) received 100 ng/ml insulin-like growth factor-I (IGF-I) for 3 h. Real-time reverse transcription/polymerase chain reaction was used to quantify the abundance of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the relative abundances of aggrecan, collagens I and II, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1). GAPDH varied zonally, but neither time nor IGF-I had an effect on it, suggesting that GAPDH is a suitable housekeeping gene for comparisons within each zone, but not across zones. IGF-I increased the expression of aggrecan and collagen II in middle/deep chondrocytes seeded for 18 h. TIMP-1 expression increased with time in control cells, suggesting that chondrocytes enter a matrix protective state after seeding. IGF-I diminished this effect, suggesting that treatment with IGF-I refocuses chondrocytes on matrix production rather than on protection from metalloproteinases. Concomitant to increasing TIMP-1, MMP-1 was detectable by 18 h in superficial cells, providing further evidence of a trend toward matrix degradation with time. Collagen I was undetected in all cells, and no differences were observed for COMP, confirming that no dedifferentiation or osteoarthritic changes occurred. Taken together, these results establish a unique understanding of individual chondrocyte behavior.

Peptidylpropyl isomerase B (PPIB): a suitable reference gene for mRNA quantification in peripheral whole blood

Quantitative real-time RT-PCR is a very powerful technique for measuring gene expression at the mRNA level. In order to compare mRNA expression in different experimental or clinical conditions, expression of a target gene has to be normalized to an appropriate internal standard, which is generally a housekeeping gene. In our study, we have tested several housekeeping genes in peripheral whole blood of healthy volunteers and patients suffering from inflammatory diseases. A first analysis of 91 samples illustrated that the mRNA expression of peptidylpropyl isomerase B (PPIB) encoding for cyclophilin B protein, is more stable than beta actin and glyceraldehyde-3-phosphate dehydrogenase, which are both commonly selected as internal standard. Among the three genes tested, beta actin displayed the highest inter-sample variation of expression. The constancy of PPIB mRNA expression was further confirmed by 214 additional samples. In conclusion, we showed that PPIB, in contrast to beta actin and glyceraldehyde-3-phosphate dehydrogenase, is a suitable housekeeping gene in human peripheral blood.

Validation of housekeeping genes for normalizing RNA expression in real-time PCR.

Analysis of RNA expression using techniques like real-time PCR has traditionally used reference or housekeeping genes to control for error between samples. This practice is being questioned as it becomes increasingly clear that some housekeeping genes may vary considerably in certain biological samples. We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis. Housekeeping genes were selected from conventionally used ones and from genes reported to be invariant in human T cell culture. None of the commonly used housekeeping genes [e.g., glyceraldehyde-phosphate-dehydrogenase (GAPDH)] were found to be suitable as internal references, as they were highly variable (>30-fold maximal variability). Furthermore, genes previously found to be invariant in human T cell culture also showed large variation in RNA expression (>34-fold maximal variability). Genes that were invariant in blood were highly variable in peripheral blood mononuclear cell culture. Our data show that RNA specifying human acidic ribosomal protein was the most suitable housekeeping gene for normalizing mRNA levels in human pulmonary tuberculosis. Validations of housekeeping genes are highly specific for a particular experimental model and are a crucial component in assessing any new model.