Expression Levels Of Housekeeping Genes Research Articles

Application of Novel Transcription Factor Machine Learning Model and Targeted Drug Combination Therapy Strategy in Triple Negative Breast Cancer.

Transcription factors (TFs) have been shown to play a key role in the occurrence and development of tumors, including triple-negative breast cancer (TNBC), with a worse prognosis. Machine learning is widely used for establishing prediction models and screening key tumor drivers. Current studies lack TF integration in TNBC, so targeted research on TF prognostic models and targeted drugs is beneficial to improve clinical translational application. The purpose of this study was to use the Least Absolute Shrinkage and Selection Operator to build a prognostic TFs model after cohort normalization based on housekeeping gene expression levels. Potential targeted drugs were then screened on the basis of molecular docking, and a multi-drug combination strategy was used for both in vivo and in vitro experimental studies. The machine learning model of TFs built by E2F8, FOXM1, and MYBL2 has broad applicability, with an AUC value of up to 0.877 at one year. As a high-risk clinical factor, its abnormal disorder may lead to upregulation of the activity of pathways related to cell proliferation. This model can also be used to predict the adverse effects of immunotherapy in patients with TNBC. Molecular docking was used to screen three drugs that target TFs: Trichostatin A (TSA), Doxorubicin (DOX), and Calcitriol. In vitro and in vivo experiments showed that TSA + DOX was able to effectively reduce DOX dosage, and TSA + DOX + Calcitriol may be able to effectively reduce the toxic side effects of DOX on the heart. In conclusion, the machine learning model based on three TFs provides new biomarkers for clinical and prognostic diagnosis of TNBC, and the combination targeted drug strategy offers a novel research perspective for TNBC treatment.

Spatial distribution of atmospheric bioaerosols in Beijing, Hangzhou and Wuhan, China

Exposure to bioaerosols can have adverse effects on human health and contribute to allergies, asthma, infectious diseases and pulmonary conditions. Bioaerosols are airborne particles of microbial, animal and plant origin. Animal- and plant-derived particles, which may disperse over long distances, are being increasingly addressed by environmental studies in addition to microorganisms. In this study, air samples were collected in various planned areas of three cities in China (Beijing, Hangzhou and Wuhan) using a new liquid-based sampler. Animal, plant and bacterial components were quantified according to the expression levels of housekeeping genes via real-time PCR and analysis of cycle threshold (Ct) values. The level of animal-derived particles in the air of Hangzhou was higher than those in the other two cities. More bacterial particles than animal or plant particles were detected in the air in Beijing compared with other cities. We also found a negative correlation between the bacterial content of bioaerosols and relative humidity and a positive correlation between bacterial content and temperature. By contrast, bioaerosol animal content was positively correlated with relative humidity and negatively correlated with temperature. In summary, high levels of bacterial particles were detected in atmospheric samples in China, although the lower concentrations of animal- and plant-derived particles should not be ignored. These data may provide important evidence for structural analyses of animal- and plant-derived bioaerosols as well as for risk assessment.

Gene Expression Correlates with the Number of Herpes Viral Genomes Initiating Infection in Single Cells.

Viral gene expression varies significantly among genetically identical cells. The sources of these variations are not well understood and have been suggested to involve both deterministic host differences and stochastic viral host interactions. For herpesviruses, only a limited number of incoming viral genomes initiate expression and replication in each infected cell. To elucidate the effect of this limited number of productively infecting genomes on viral gene expression in single cells, we constructed a set of fluorescence-expressing genetically tagged herpes recombinants. The number of different barcodes originating from a single cell is a good representative of the number of incoming viral genomes replicating (NOIVGR) in that cell. We identified a positive correlation between the NOIVGR and viral gene expression, as measured by the fluorescent protein expressed from the viral genome. This correlation was identified in three distinct cell-types, although the average NOIVGR per cell differed among these cell-types. Among clonal single cells, high housekeeping gene expression levels are not supportive of high viral gene expression, suggesting specific host determinants effecting viral infection. We developed a model to predict NOIVGR from cellular parameters, which supports the notion that viral gene expression is tightly linked to the NOIVGR in single-cells. Our results support the hypothesis that the stochastic nature of viral infection and host cell determinants contribute together to the variability observed among infected cells.

Non-targeted effects of virus-induced gene silencing vectors on host endogenous gene expression.

Virus-induced gene silencing (VIGS) uses recombinant viruses to study gene function; however, the effect of the virus vector itself on the gene expression of the host is not always considered. In our work, we investigated non-targeted gene expression changes of the host in order to see how often these changes appear. Effects of various VIGS vector infections were analysed by monitoring gene expression levels of housekeeping genes by Northern blot analysis in four different hosts. We found that non-targeted changes happens very often. More importantly, these non-targeted effects can cause drastic changes in the gene-expression pattern of host genes that are usually used as references in these studies. We have also found that in a tobacco rattle virus (TRV)-based VIGS, the presence of foreign sequences in the cloning site of the vector can also have a non-targeted effect, and even the use of an internal control can lead to unpredicted changes. Our results show that although VIGS is a very powerful technique, the VIGS vector, as a pathogen of the host, can cause unwanted changes in its gene-expression pattern, highlighting the importance of careful selection of both the genes to be tested and those to be used as references in the planned experiments.

Regression hidden Markov modeling reveals heterogeneous gene expression regulation: a case study in mouse embryonic stem cells.

BackgroundStudies have shown the strong association between histone modification levels and gene expression levels. The detailed relationships between the two can vary substantially due to differential regulation, and hence a simple regression model may not be adequate. We apply a regression hidden Markov model (regHMM) to further investigate the potential multiple relationships between genes and histone methylation levels in mouse embryonic stem cells.ResultsSeven histone methylation levels are used in the study. Averaged histone modifications over non-overlapping 200 bp windows on the range transcription starting site (TSS) ± 1 Kb are used as predictors, and in total 70 explanatory variables are generated. Based on regHMM results, genes segregated into two groups, referred to as State 1 and State 2, have distinct association strengths. Genes in State 1 are better explained by histone methylation levels with R2=.72 while those in State 2 have weaker association strength with R2=.38. The regression coefficients in the two states are not very different in magnitude except in the intercept,.25 and 1.15 for State 1 and State 2, respectively. We found specific GO categories that may be attributed to the different relationships. The GO categories more frequently observed in State 2 match those of housekeeping genes, such as cytoplasm, nucleus, and protein binding. In addition, the housekeeping gene expression levels are significantly less explained by histone methylation in mouse embryonic stem cells, which is consistent with the constitutive expression patterns that would be expected.ConclusionGene expression levels are not universally affected by histone methylation levels, and the relationships between the two differ by the gene functions. The expression levels of the genes that perform the most common housekeeping genes’ GO categories are less strongly associated with histone methylation levels. We suspect that additional biological factors may also be strongly associated with the gene expression levels in State 2. We discover that the effect of the presence of CpG island in TSS ± 1 Kb is larger in State 2.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-360) contains supplementary material, which is available to authorized users.

Validation of candidate reference genes in Bifidobacterium adolescentis for gene expression normalization

Normalization is an essential prerequisite for producing accurate real-time PCR expression analyses. The objective of this study is the selection of a set of optimal reference genes in Bifidobacterium adolescentis gene expression studies under bile exposure. B adolescentis is a particularly abundant species in the human adults gut microbiota, exerting relevant probiotic activities. In the gastrointestinal tract, bile represents a hard challenge for bacterial survival, because of its toxic effect. The natural exposure to bile in the colonic environment induces cells adaptation and tolerance mechanisms in bifidobacteria, which determines changes in gene expression profile, influencing the expression levels of housekeeping genes. In this context, the stability of 9 putative reference genes (cysS, purB, recA, rpoB-L, GADPH-R, 16S rRNA, glnA1, gyrA2, sdhA) was examined in B. adolescentis exposed to bile extract, using two different software (BestKeeper and NormFinder). Both algorithms identified gyrA2 and sdhA as the most stable genes under our experimental conditions, while 16S rRNA is the least reliable HKGs. To our best knowledge, this is the first attempt to validate reference genes in Bifidobacterium spp. and the results offer an appropriate set of reference genes suitable for qRT-PCR studies on B. adolescentis strains under bile stress.

Selection and validation of reference genes for real-time qRT-PCR normalization in different tissues of Eucalyptus tereticornis

AbstractReference genes are generally used as endogenous normalization factor for relative quantification of target genes in quantitative real-time PCR (qRT-PCR). The present work aimed at identifying suitable reference genes for normalization of qRT-PCR data in tissues of Eucalyptus tereticornis. The expression levels of housekeeping genes like Actin (EtAct2), Isocitrate dehy - drogenase (EtIDH), ribosomal RNA (Et18s rRNA), SAND family protein (EtSAND), Histone protein (EtH2B), α-Tubulin (EtTUB), and eukaryotic initiation factor (EteIF4B) were studied to characterize their normalization stability in different tissues including young leaves, internodes, developing and mature xylem. The expression level of these genes was analyzed using different algorithms like geNorm, NormFinder and Best- Keeper. Among the seven reference genes analyzed, EtAct2 was expressed with less variance and was found to be the most stable reference gene across different tissues using all the three programs, while the least stable gene identified was EtH2B. Further, the normalization efficiency of the reference genes were assessed to predict the expression levels of three primary cell wall specific cellulose synthase transcripts (EtCesAs) in E. tereticornis tissues. The relative expression of EtCesA4, EtCesA5 and EtCesA6 was determined to be 3-19 fold higher in leaf and internode tissues when compared to developing and mature xylem tissues. This study will allow accurate normalization of qRT-PCR experiments across different tissues in E. tereticornis for future genomic research in this tropical eucalypt species.

Determination of an Effective Housekeeping Gene for the Quantification of mRNA for Forensic Applications*

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood.

Preservation of Ranking Order in the Expression of Human Housekeeping Genes

Housekeeping (HK) genes fulfill the basic needs for a cell to survive and function properly. Their ubiquitous expression, originally thought to be constant, can vary from tissue to tissue, but this variation remains largely uncharacterized and it could not be explained by previously identified properties of HK genes such as short gene length and high GC content. By analyzing microarray expression data for human genes, we uncovered a previously unnoted characteristic of HK gene expression, namely that the ranking order of their expression levels tends to be preserved from one tissue to another. Further analysis by tensor product decomposition and pathway stratification identified three main factors of the observed ranking preservation, namely that, compared to those of non-HK (NHK) genes, the expression levels of HK genes show a greater degree of dispersion (less overlap), stableness (a smaller variation in expression between tissues), and correlation of expression. Our results shed light on regulatory mechanisms of HK gene expression that are probably different for different HK genes or pathways, but are consistent and coordinated in different tissues.

Abstract 1576: Prostaglandin I2 signaling regulates micro-metastasis in lung cancer

Abstract Background: Bone marrow-derived cells (BMDCs) play an important role in the initiation of neo-angiogenesis, and contribute to the diseases including ischemic diseases, retinopathy, and cancer. We have reported that BMDCs express prostaglandin I2 (PGI2) specific receptor, IP and the PGI2-IP signaling system maintains the function of BMDCs. Additionally, we have reported that the PGI2-IP signaling in BMDCs play an important role for tumor growth, by using mouse bone marrow transplanted models and tumor xenograft mouse models. Objectives: The purpose of this study was to test the hypothesis that PGI2-IP signaling system regulates micro-metastasis in lung cancer. Methods: In vitro study, 5-bromo-2’-deoxy-uridine (BrdU) cell proliferation assay and clonogenic cell assay were used to clarify the effect on lung cancer cell (A549, PC9) proliferation and cellular form in the existence of IP receptor agonist or antagonist. Human cytokine antibody array was performed to detect the expression of angiogenic cytokines and growth factors secreted by the treated cells. In vivo study, we employed both a mouse lung metastasis model and a mouse bone marrow transplantation model. We used GFP-labeled or DsRed-labeled lung cancer cells to distinguish cancer cells from BMDCs. Lung cancer cells were injected into the tail vein of mice (ICR nu/nu), which were transplanted with bone marrow cells from GFP-labeled wild-type or IP knockout mice. Real-time RT-PCR was used to quantify lung metastasis of human cancer cells in nude mice, and the expression levels of housekeeping genes (human and mouse GAPDH) in the lungs were measured. Immunofluorescence assay was performed to evaluate angiogenesis in pulmonary metastatic tumors. Results: In vitro study, PGI2-IP signaling didn't impair proliferation of lung cancer cells and production of angiogenic cytokines secreted by tumors. In the mice lung metastasis model, micro-metastasis in lung cancer was regulated by PGI2-IP signaling system. RT-PCR assay showed that PGI2-IP signaling changed the expression levels of human lung cancer cells in mice lungs. In the immunofluorescence assay, PGI2-IP signaling influenced the expression of pericyte markers around the tumors. Conclusions: The present study demonstrated that PGI2-IP signaling system regulates micro-metastasis in lung cancer. These results suggest that PGI2-IP system may become a novel therapeutic target in lung metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2011-1576

Housekeeping Gene Transcript Abundance in Bovine Fertilized and Cloned Embryos

The objective of this study was to compare housekeeping gene expression levels, relative to total mRNA, across different stages of bovine preimplantation development in embryos generated by IVF and somatic cell nuclear transfer (SCNT). We first analyzed the levels of total RNA recovered from different stages of preimplantation development. A similar RNA level was observed from oocytes to 16-cell stage embryos with a significant increase at morula and blastocyst stages. Then we used an absolute mRNA determination method that accounts for the RNA level in the embryo by quantifying copies of transcripts normalized to loaded cDNA amount. The number of housekeeping genes mRNA copies per nanogram of cDNA was compared among samples obtained from different stages of preimplantation IVF-derived embryos. None of the genes analyzed (GAPDH, PPIA, ACTB, RPL15, GUSB, and Histone H2A.2) maintained constant levels throughout preimplantation development, indicating that they are not suitable for normalizing gene expression across developmental stages. We then compared expression of housekeeping genes between IVF and SCNT embryos at different embryonic stages. We found different levels of transcript abundance between IVF and SCNT embryos for GAPDH, RPL15, GUSB, and ACTB. On the other hand, Histone H2A.2 and PPIA were similar between IVF and SCNT embryos at each stage analyzed, although they varied across stages as previously mentioned.

194 HOUSEKEEPING GENE EXPRESSION OF BOVINE FERTILIZED AND CLONED EMBRYOS

Real-time RT-PCR can accurately quantify mRNA levels in pre-implantation embryos; however, comparisons among different embryonic stages and among embryos produced by different means often rely on a control gene, which is commonly assumed to remain constant across samples. The objective of this study was to compare housekeeping gene expression levels, relative to total mRNA, across different stages of bovine pre-implantation development in embryos generated by IVF and somatic cell nuclear transfer (SCNT). Embryos were produced according to standard protocols (Ross et al. 2006 Biotechniques 41, 741–750). Total RNA was collected from 3 pools of 10 oocyte/embryos at metaphase II (MII), PN, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages and reverse-transcribed using oligo-dT primers. The cDNA was then amplified using PCR (Kocabas et al. 2006 PNAS 103, 14 027–14 032). All amplified cDNA samples were diluted to 1 ng μL–1, as determined by NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and corroborated using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). For real-time PCR, 2 μL of cDNA was analyzed in duplicates. Absolute quantification was performed as previously described (Iager et al. 2008 Cloning Stem Cells doi:10.1089), using SYBR-green chemistry and standard curves specific for each gene. The number of RNA copies per nanogram of amplified cDNA was compared among samples using ANOVA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin A, β-actin, ribosomal protein L-15 (RPL-15), and β-glucuronidase (GUS) expression levels were similar in MII oocyte, 1-, 2-, 4-, and 8-cell embryos, while a significant increase at morula and blastocyst stages was observed (P < 0.05). A similar pattern of expression was observed for 18S ribosomal RNA, but with a significant decrease from morula to blastocyst stages (P < 0.05). Histone H2A expression was significantly higher at 1-cell stage, similar from 2-cell to morula stages and lowest at the blastocyst stage. We then compared expression between IVF and SCNT embryos at 2-, 4-, and 8-cell and blastocyst stages. GAPDH, RPL-15, GUS, and β-actin were significantly different among groups in at least 3 of the analyzed stages, which in all cases included blastocysts. 18s-rRNA was different among IVF and SCNT embryos only at the 8-cell stage, while no differences were observed at any stages for histone H2A and cyclophilin A. At the blastocyst stage, the lowest overall variability among IVF and SCNT embryos was observed for 18s-rRNA. In conclusion, none of the evaluated housekeeping genes showed consistent mRNA expression levels across developmental stages of IVF embryos. In addition, SCNT embryos, compared to IVF, had different levels of gene expression for commonly used housekeeping genes, which, if neglected, might result in data misinterpretation. In our conditions, histone H2A had similar expression levels between IVF and SCNT embryos across different stages and showed less variability than cyclophilin A. Finally, for comparisons at the blastocyst stage, 18s-rRNA had the least variability among IVF and SCNT embryos.

Constitutive mRNA expression of DNA repair-related genes as a biomarker for clinical radio-resistance: A pilot study in prostate cancer patients receiving radiotherapy

Purpose: Repair of radiation-induced DNA damage is believed to play a critical role in the development of adverse reactions in radiotherapy patients. Constitutive mRNA expression of repair genes was investigated in such patients to analyze whether expression patterns are predictive for therapy-related acute side effects.Materials and methods: Prostate cancer patients (n = 406) receiving intensity-modulated radiotherapy were recruited in a prospective epidemiological study. Adverse effects were monitored during therapy using common toxicity criteria. For expression analyses, samples from 58 patients were selected according to their observed grade of clinical side effects to radiotherapy. Expression profiles were generated from peripheral blood lymphocytes using customized cDNA-arrays which carried probes for 143 DNA repair or repair-related genes. In addition, expression of selected genes was confirmed by quantitative real-time reverse transcription PCR (RT-PCR). Constitutive mRNA expression profiles were analyzed for predicting acute clinical radiosensitivity or radio-resistance.Results: Cluster analysis identified 19 differentially expressed genes. Many of these genes are involved in DNA double strand break repair. Expression levels of these genes differed up to 7-fold from the mean of all patients whereas expression levels of housekeeping genes varied only up to 2-fold. High expression of the identified genes was associated with a lack of clinical radiation sensitivity thus indicating radio-resistance.Conclusions: Constitutive expression of DNA repair-related genes may affect the development of acute side effects in radiotherapy patients, and high expression levels of these genes seem to support protection from adverse reactions.

Real-Time PCR Gene Expression Profiling of Microarray Gene Signatures in Acute Leukemia.

Cancer subtype discovery and classification using microarray gene signatures has the potential to transform pathological diagnosis but measurement of indicator genes in routine practice remains difficult. We tested use of real-time PCR measurement of indicator genes for AML and ALL (Golub et al, Science, 1999) as a method for validation and application of microarray gene signatures. Mononuclear cells (MC) were isolated from whole bone marrow (BM) aspirates by density gradient centrifugation and sorted into unselected (total), CD34+ve and CD34-ve fractions. The mRNA in each fraction was globally amplified using a PolyA PCR method. We measured the expression profile of the 17 top ranked genes (cystatin C, leptin receptor, fumarylacetoacetate, CD33, HoxA9, adipsin, proteoglycan 1, LTC4 synthase, LYN, C-myb, MB-1, cyclin D3, SNF2, RbAp48, proteasome iota, HkrT-1 and E2A) from Golub et al (1999) by real-time PCR. All values were calibrated against control standards and normalized to the mean of three housekeeping genes (IF2-beta, GAPDH and human ribosomal protein S9). Data for all 17 genes were obtained for 4 (ALL), 26 (AML), 12 (AML remission) and 9 (morphologically normal) BM samples, each fractionated into three fractions (total MC, CD34+ve MC & CD34−ve MC). There was no significant difference in the mean of three housekeeping gene expression levels between the diagnostic groups. Comparison of the expression level of the other genes confirmed ability to separate AML and ALL, whilst the direction of expression change (increased or decreased) for each gene between AML and ALL was the same as found by Golub et al. In particular, c-myb showed largest significant increase in ALL vs AML in the total BM fraction, whilst cystain c was increased in AML in the CD34−ve fraction. hSNF2b was significantly increased in the ALL total B.M fraction and Hox-A9 was significantly increased in the AML CD34+ve B.M fraction. Furthermore expression level of LYN and CD33 was significantly increased in AML compared to remission AML, indicating ability of the method to determine activity status of disease. In addition, several of the genes provided better separation between AML and ALL when measured in the CD34+ve and −ve fractions indicating more prominent expression in cells of different maturity and that prior fractionation is diagnostically more informative. The results demonstrate ability of the method to validate gene expression signatures by an independent method, which is simple, sensitive and robust, allowing translation to routine clinical use. Whilst the present study used AML and ALL, in principle the method could be extended to any other tumor type for which gene signatures exist.

CD34+ Cell Selection Is Required to Accurately Measure HOXA9 Levels by Quantitative RT-PCR in Patients with Myelodysplastic Syndrome.

In recent years, it has become increasingly clear that overexpression of specific major HOX genes is linked to transformation of the malignant hematopoietic stem cells underlying human acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). For example, several recent studies indicate that overexpression of HOXA9 is associated with a poor outcome in AML and MDS. As we initiated our own studies of HOXA9 expression in MDS, we were concerned about the validity of studying whole bone marrow (BM) cell RNA obtained at diagnosis, due to the diversity of cell types and low blast cell numbers in the BM that characterize MDS, and prior studies of G. Savageau and K. Humphries demonstrating that HOXA9 expression in normal BM cells is limited to the CD34+ fraction, which is enriched for hematopoietic stem and progenitor cells. Thus, we initiated this study to investigate whether valid HOXA9 expression levels can be determined from whole BM cell RNAs of MDS patients or whether CD34+ cell separation is required prior to analysis. BM aspirate samples were collected at diagnosis from 9 patients with MDS and subjected to Ficoll-Hypaque light density cell separation, followed by magnetic bead separation to remove CD3+/CD19+ mature lymphoid cells. The CD3−/CD19− fraction was further fractionated into CD34+ and CD34− populations. RNA was extracted by standard methods and two-step quantitative real-time RT-PCR was performed using SYBR Green to measure HOXA9 and control housekeeping gene expression levels. RNAs from peripheral blood CD34+ cells from 8 healthy donors served as controls. Relative HOXA9 expression levels were obtained by normalization to three housekeeping genes (GAPDH, YWHAZ, and RPL4) for each fraction. Our results conclusively show that HOXA9 levels of CD34+ cells from MDS patients are on average three-fold higher than those of CD34− cells from the same patient (p=0.004, Wilcoxon signed rank). Thus, as is the case for normal BM cells, HOXA9 expression in MDS is highest in the stem and progenitor cell fraction. Our results indicate that CD34+ cell selection must be carried out to accurately assess the expression of HOXA9 in the malignant clone of MDS patients. Unselected BM cell measurements include variable fractions of CD34− and CD34+ cells, which can have significant variability, even in samples obtained from the same patient, thus confounding attempts to accurately measure HOXA9 levels and determine the contribution of this oncogene to MDS. Moreover, we detected levels of HOXA9 expression in the CD34+ cells of MDS patients that were on average 4.3 times those of normal controls (p=0.004, Wilcoxon rank sum). Our studies indicate that in order to advance understanding of the molecular pathogenesis of MDS, the expression levels of specific HOX genes and their co-regulators will need to be carefully evaluated in purified CD34+ BM cells from patients with this disease.

Quantifying Aotus monkey cytokines by real-time quantitative RT-PCR

Aotus spp. monkeys are considered the ideal model for studying the progress of malarial infection and the immune response it elicits. We describe the use of a recently developed technique, real-time quantitative RT-PCR, to quantify several Aotus monkey cytokine mRNAs involved in Th1/Th2 responses (IL-4, IL-10, TNF-β and IFN-γ). Specific primers were designed for each cytokine and standard curves were constructed using serial dilutions of pDNA containing each target sequence. Results were normalized to GAPDH housekeeping gene expression levels. Standard curves showed high correlation coefficients and were linear over a wide range of copy numbers. Quantification of Aotus samples showed little intra- and inter-experiment variation, thus, the technique has proven to be highly reproducible and sensitive allowing us to detect as little as 25 copies/μl of target DNA. This technique will allow studying Th1 and Th2 cytokine patterns elicited in response to infection for prospectively evaluating the efficacy of malarial vaccines.

A Partial Transcriptome of Human Epidermis

Serial analysis of gene expression (SAGE) is a powerful technique for global expression profiling without prior knowledge of the genes of interest. We carried out SAGE analysis of purified keratinocytes derived from human skin biopsy specimens, resulting in a partial transcriptome of human epidermis. We identified 7645 unique SAGE tags with quantitative information from 15,131 collected SAGE tags obtained from ∼3×106 epidermal cells. This catalog contains a large number of genes that were not previously known to be expressed by human epidermis. Comparison with the databases of all known human SAGE tags allowed us to identify a number of keratinocyte-specific tags that putatively correspond to formerly unknown genes. Surprisingly, human epidermal keratinocytes in vivo show relatively low expression levels of genes typically associated with epidermal differentiation, whereas the expression levels of housekeeping genes are considerably higher than in cultured keratinocytes. This study provides a first step toward a transcriptome of human epidermis and, as such, harbors a wealth of information to identify genes involved in skin function, and candidate genes for genetic skin disorders.

Response to 'Lymphoid organs contain diverse cells expressing self-molecules'

Pietropaolo et al. and Pugliese et al. are correct in arguing that our study does not entirely rule out the possibility that other thymic cells can express a diverse range of tissue-specific antigens. It was neither our intention, nor do we consider it appropriate, to address the issue of gene expression in a categorical yes-or-no fashion. It has long been known that a certain degree of so-called illegitimate transcription allows for the presence of one or few transcripts of any given gene in any given cell1, 2. Consequently, the detection of a specific transcript reflects, to a large extent, the sensitivity of the method applied, and the designation of a positive signal has to be defined in the particular context. We compared expression of tissue-specific genes in five different thymic cell types by carefully calibrating the amount of input cDNA according to the expression levels of housekeeping genes. The PCR data thus allow for an estimate of the relative abundance of transcripts in the respective cell types. The central observation of our study is that mTECs are clearly distinct in the strength as well as the diversity of genes that are promiscuously expressed. We have, in the meantime, extended this observation to mTECs isolated from the human thymus (B. K. et al. unpublished data).