Orthogonal control of mean and variability of endogenous genes in a human cell line.
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ABSTRACT:
SUBMITTER: Bonny AR
PROVIDER: S-EPMC7804932 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:While gene expression noise has been shown to drive dramatic phenotypic variations, the molecular basis for this variability in mammalian systems is not well understood. Gene expression has been shown to be regulated by promoter architecture and the associated chromatin environment. However, the exact contribution of these two factors in regulating expression noise has not been explored. Using a dual-reporter lentiviral model system, we deconvolved the influence of the promoter sequence to systematically study the contribution of the chromatin environment at different genomic locations in regulating expression noise. By integrating a large-scale analysis to quantify mRNA levels by smFISH and protein levels by flow cytometry in single cells, we found that mean expression and noise are uncorrelated across genomic locations. Furthermore, we showed that this independence could be explained by the orthogonal control of mean expression by the transcript burst size and noise by the burst frequency. Finally, we showed that genomic locations displaying higher expression noise are associated with more repressed chromatin, thereby indicating the contribution of the chromatin environment in regulating expression noise.
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Project description:In human glioma, quantitative real-time reverse-transcription PCR (qPCR) is a frequently used research tool. However, no systematic analysis of suitable reference genes for reliable gene expression analysis has been performed so far. In the current study, we tested 19 commonly used reference genes for their expression stability in human astrocytoma WHO Grade II, astrocytoma WHO Grade III, and glioblastoma (WHO Grade IV) both alone and compared with normal brain. First, equivalence tests for equal expression of candidate genes were applied, and those genes showing differential expression were ruled out from further analyses. Second, expression stability of the remaining candidate genes was determined by the NormFinder software. Generally, glioblastoma exhibited the highest expression levels and largest variability of candidate genes, whereas the opposite was true for normal brain. Even though Normfinder analyses revealed a large number of genes suitable for normalization in each of the tumor subgroups and across these groups, this number was drastically reduced after inclusion of normal brain into the analyses: Only GAPDH, IPO8, RPL13A, SDHA, and TBP were expected not to be differentially expressed; NormFinder analysis indicated favorable stability values for all of these genes, with TBP and IPO8 being the most stable ones. These 5 genes represent different physiological pathways and may be regarded as universal reference genes applicable for accurate normalization of gene expression in human astrocytomas of different grades (WHO Grades II-IV) alone and compared with normal brain, thereby enabling longitudinally designed studies (eg, in astrocytoma before and after malignant transformation).
| S-EPMC2940642 | biostudies-literature
Project description:PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18S<TBP<B2M<PGK1<HPRT1<GUSB<GAPDH<PPIA-RPLP0. In this study, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable. CONCLUSION: This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use of PPIA-RPLP0 ECGs pair in quantitative reverse transcription PCR across the OS epithelium.
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Project description:Human endogenous retroviruses (HERVs) integrated into the human genome millions of years ago, and their expression has been described both in healthy tissues and in pathology. In the present study, we study the differential expression of multiple HERV families in the presence of human immunodeficiency virus (HIV) infection in the peripheral blood mononuclear cells (PBMCs) of HIV-positive individuals, in HIV-infected cell lines, and in vitro HIV-infected macrophages/monocytes of healthy donors, utilizing publicly available data sets for secondary analysis using a pipeline appropriate for HERV transcription detection and comparison. We recognized significantly decreased expression of HERV-H in treatment-naïve HIV patients with viremia and increased expression of multiple HERV families in the PBMCs of HIV-infected individuals under combination antiretroviral therapy compared to healthy donors, in in vitro HIV-infected Stanford University pediatric T (SUP-T1) cells at 24 hours after HIV infection compared to non-infected SUP-T1 cells, and in in vitro HIV-infected MT4 T cells at 3 days after HIV infection compared to non-infected MT4 T cells. We recognized significantly increased expression of HERV-W, after HIV infection in monocyte-derived macrophage cell culture; no difference was found in HERV expression after HIV infection of monocyte cultures. We found a significantly decreased expression of HERV-K (HML-6) in donor-derived macrophage cultures at 36 hours after HIV infection, which does not persist at 6 days after infection. HIV infection modifies the expression of multiple HERV families in the PBMCs of HIV patients and in T cell-derived cultures, while the effect of HIV infection on HERV expression appears to be more restricted in cells of monocytic origin.IMPORTANCEIn this work, we demonstrated that human immunodeficiency virus (HIV) infection leads to the modification of the human endogenous retrovirus (HERV) expression. Differential expression of multiple HERVs was found in peripheral blood mononuclear cells derived from HIV-infected patients compared to healthy donors and HIV-infected T cell cultures compared to non-infected. The effect of HIV presence on HERV expression appears to be more restricted in cells of monocytic origin, as only deregulation of HERV-W and HERV-K (HML-6) was found in these cell cultures after their infection with HIV. Multiple factors contribute to this aberrant HERV expression, and its levels appear to be modified in a time-dependent manner. Further studies and the development of optimized in vitro protocols are warranted to elucidate the interactions between HIV and HERVs in detail.
| S-EPMC10715072 | biostudies-literature
Project description:BackgroundGene expression microarrays and real-time PCR are common methods used to measure mRNA levels. Each method has a fundamentally different approach of normalization between samples. Relative quantification of gene expression using real-time PCR is often done using the 2(/\)(-DeltaDeltaCt) method, in which the normalization is performed using one or more endogenous control genes. The choice of endogenous control gene is often arbitrary or bound by tradition. We here present an analysis of the differences in expression results obtained with microarray and real-time PCR, dependent on different choices of endogenous control genes.ResultsIn complex tissue, microarray data and real-time PCR data show the best correlation when endogenous control genes are omitted and the normalization is done relative to total RNA mass, as measured before reverse transcription.ConclusionWe have found that for real-time PCR in heterogeneous tissue samples, it may be a better choice to normalize real-time PCR Ct values to the carefully measured mass of total RNA than to use endogenous control genes. We base this conclusion on the fact that total RNA mass normalization of real-time PCR data shows better correlation to microarray data. Because microarray data use a different normalization approach based on a larger part of the transcriptome, we conclude that omitting endogenous control genes will give measurements more in accordance with actual concentrations.
| S-EPMC2779820 | biostudies-literature
Project description:The discovery of microRNAs (miRNAs) added an extra level of intricacy to the already complex system regulating gene expression. These single-stranded RNA molecules, 18-25 nucleotides in length, negatively regulate gene expression through translational inhibition or mRNA cleavage. The discovery that aberrant expression of specific miRNAs contributes to human disease has fueled much interest in profiling the expression of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalised to an endogenous control (EC) gene, which ideally, is stably-expressed across the test sample set. A universal endogenous control suitable for every tissue type, treatment and disease stage has not been identified and is unlikely to exist, so, to avoid introducing further error in the quantification of expression data it is necessary that candidate ECs be validated in the samples of interest. While ECs have been validated for quantification of mRNA expression in various experimental settings, to date there is no report of the validation of miRNA ECs for expression profiling in breast tissue. In this study, the expression of five miRNA genes (let-7a, miR-10b, miR-16, miR-21 and miR-26b) and three small nucleolar RNA genes (RNU19, RNU48 and Z30) was examined across malignant, benign and normal breast tissues to determine the most appropriate normalisation strategy. This is the first study to identify reliable ECs for analysis of miRNA by RQ-PCR in human breast tissue.
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Project description:OBJECTIVE(S):Hypoxia is a serious challenge for treatment of solid tumors. This condition has been manifested to exert significant therapeutic effects on glioblastoma multiform or (WHO) astrocytoma grade IV. Hypoxia contributes numerous changes in cellular mechanisms such as angiogenesis, metastasis and apoptosis evasion. Furthermore, in molecular level, hypoxia can cause induction of DNA breaks in tumor cells. Identification of mechanisms responsible for these effects can lead to designing more efficient therapeutic strategies against tumor progression which results in improvement of patient prognosis. Materials and Methods : In order to identify more hypoxia regulated genes which may have a role in glioblastoma progression, cDNA-AFLP was optimized as a Differential display method which is able to identify and isolate transcripts with no prior sequence knowledge. RESULTS:Using this method, the current study identified 120 Transcription Derived Fragments (TDFs) which were completely differentially regulated in response to hypoxia. By sequence homology searching, the current study could detect 22 completely differentially regulated known genes and two unknown sequence matching with two chromosome contig and four sequence matches with some Expressed Sequence Tags (ESTs). CONCLUSION:Further characterizing of these genes may help to achieve better understanding of hypoxia mediated phenotype change in tumor cells.
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Project description:We have examined the human mammary tumor cell line T47D and have found that these cells produce virus-like particles which band at the typical density for retroviral particles on a sucrose gradient, possess reverse transcriptase activity, and package HERV-K10-like sequences. Using this information and a bacterial expression system to identify long open reading frames, we have identified individual clones which have full-length open reading frames for reverse transcriptase and RNase H and which could encode the reverse transcriptase activity detected in these cells.
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