Transcription profiling of human samples differing in hemoglobin transcripts to investigate cross-hybridization comparison between cRNA target and cDNA target
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Aron Eklund
PROVIDER: E-GEOD-4532 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We used two RNA samples that differed only by a set of three hemoglobin transcripts to compare microarray cross-hybridization between two target preparation protocols. We found widespread cross-hybridization using standard cRNA target but substantially less cross-hybridization using cDNA target. Keywords: protocol comparison
2006-09-08 | GSE4532 | GEO
Project description:cDNA microarrays have become the technology of choice for profiling the expression of thousands of genes simultaneously in a single sample. However, cross-hybridization between targets and probes that do not correspond must be considered carefully during the design, analysis, and interpretation of these experiments. Here, we examined this potentially confounding issue by competitively hybridizing fragments with similar sequence obtained from two paralogous genes to a custom cDNA microarray that contains probes for only one of these two genes. We found that although a large contiguous portion of these two fragments was 84% identical and only one target was represented on the array, there was very little cross-hybridization of the non-represented target to either the paralogous probes or other (non-similar) features. We conclude that cDNA microarrays are indeed very specific and that promiscuous paralog hybridization adds little noise to the data. More generally, the absence of specific probes for any given gene does not increase the chance for cross-hybridization between non-specific probes and targets, even if sequence similarity is relatively high.
2009-07-03 | GSE16939 | GEO
Project description:BACKGROUND & AIMS: Recent studies revealed that hemoglobin is expressed in some non-erythrocytes and it suppresses oxidative stress when overexpressed. Oxidative stress plays a critical role in the pathogenesis of non-alcoholic steatohepatitis (NASH). This study was to investigate whether hemoglobin is expressed in hepatocytes and how it is related to oxidative stress in NASH patients. METHODS: Microarray was performed to identify differentially expressed genes in NASH. Quantitative real time PCR (qRT-PCR) was used to examine gene expression levels. Western blotting and immunofluorescence staining were employed to examine hemoglobin proteins. Flow cytometry was used to measure intracellular oxidative stress. RESULTS: Analysis of microarray gene expression data has revealed a significant increase in the expression of hemoglobin alpha (HBA1) and beta (HBB) in liver biopspies from NASH patients. Increased hemoglobin expression in NASH was validated by qRT-PCR. However, the expression of erythrocyte specific marker genes such as SPTA, SPTB, GYPA, GATA1, and ALAS2 did not change, indicating that increased hemoglobin expression in NASH was not from erythropoiesis, but could result from increased expression in hepatocytes. Immunofluorescence staining demonstrated positive HBA1 and HBB expression in the hepatocytes of NASH livers. Hemoglobin expression was also observed in human hepatocellular carcinoma HepG2 cell line. Furthermore, treatment with hydrogen peroxide, a known oxidative stress inducer, induced a dose dependent increase in HBA1 expression in HepG2 cells. Intriguingly, forced hemoglobin expression suppressed oxidative stress. CONCLUSIONS: Oxidative stress upregulates hemoglobin expression in hepatocytes. Suppression of oxidative stress by hemoglobin could be a mechanism to protect hepatocytes from oxidative damage. These findings suggest that hemoglobin is an inducible antioxidant in hepatocytes in response to increased oxidative stress as found in NASH livers. Twelve biopsy diagnosed NASH patients were included in this study. For control groups, total RNA from 5 different subjects were purchased from ADMET. These subjects are free from liver disease.
2010-10-20 | E-GEOD-24807 | biostudies-arrayexpress
Project description:Through high throughput compound screening, we've identified compounds that induce the expression of fetal hemoglobin. This study contains CUT&RUN data of a novel target, WIZ.
2024-06-19 | E-MTAB-13890 | biostudies-arrayexpress
Project description:Plasmodium falciparum, the causative agent of malaria, continues to remain a global health threat since these parasites are now resistant to all anti-malaria drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the hosts main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here we utilize both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that our inhibitor is on-target for PfA-M17 and has the ability to kill parasites at nanomolar concentrations. Thus, in contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.
2022-09-12 | PXD032358 | Pride
Project description:BACKGROUND & AIMS: Recent studies revealed that hemoglobin is expressed in some non-erythrocytes and it suppresses oxidative stress when overexpressed. Oxidative stress plays a critical role in the pathogenesis of non-alcoholic steatohepatitis (NASH). This study was to investigate whether hemoglobin is expressed in hepatocytes and how it is related to oxidative stress in NASH patients. METHODS: Microarray was performed to identify differentially expressed genes in NASH. Quantitative real time PCR (qRT-PCR) was used to examine gene expression levels. Western blotting and immunofluorescence staining were employed to examine hemoglobin proteins. Flow cytometry was used to measure intracellular oxidative stress. RESULTS: Analysis of microarray gene expression data has revealed a significant increase in the expression of hemoglobin alpha (HBA1) and beta (HBB) in liver biopspies from NASH patients. Increased hemoglobin expression in NASH was validated by qRT-PCR. However, the expression of erythrocyte specific marker genes such as SPTA, SPTB, GYPA, GATA1, and ALAS2 did not change, indicating that increased hemoglobin expression in NASH was not from erythropoiesis, but could result from increased expression in hepatocytes. Immunofluorescence staining demonstrated positive HBA1 and HBB expression in the hepatocytes of NASH livers. Hemoglobin expression was also observed in human hepatocellular carcinoma HepG2 cell line. Furthermore, treatment with hydrogen peroxide, a known oxidative stress inducer, induced a dose dependent increase in HBA1 expression in HepG2 cells. Intriguingly, forced hemoglobin expression suppressed oxidative stress. CONCLUSIONS: Oxidative stress upregulates hemoglobin expression in hepatocytes. Suppression of oxidative stress by hemoglobin could be a mechanism to protect hepatocytes from oxidative damage. These findings suggest that hemoglobin is an inducible antioxidant in hepatocytes in response to increased oxidative stress as found in NASH livers.
2010-10-21 | GSE24807 | GEO
Project description:cDNA microarrays have become the technology of choice for profiling the expression of thousands of genes simultaneously in a single sample. However, cross-hybridization between targets and probes that do not correspond must be considered carefully during the design, analysis, and interpretation of these experiments. Here, we examined this potentially confounding issue by competitively hybridizing fragments with similar sequence obtained from two paralogous genes to a custom cDNA microarray that contains probes for only one of these two genes. We found that although a large contiguous portion of these two fragments was 84% identical and only one target was represented on the array, there was very little cross-hybridization of the non-represented target to either the paralogous probes or other (non-similar) features. We conclude that cDNA microarrays are indeed very specific and that promiscuous paralog hybridization adds little noise to the data. More generally, the absence of specific probes for any given gene does not increase the chance for cross-hybridization between non-specific probes and targets, even if sequence similarity is relatively high. PCR products from two paralogs (arginine vasotocin/vasopressin and isotocin/mesotocin/oxytocin) were competitively hybridized onto a glass cDNA microarray platform of the same species. The platform contained probes for only one of the two paralogs in an effort to assess cross-hybridization between paralogous targets and probes.
2010-05-16 | E-GEOD-16939 | biostudies-arrayexpress
Project description:Cross-hybridization comparison between cRNA target and cDNA target
| PRJNA94457 | ENA
Project description:Through high throughput compound screening, we've identified compounds that induce the expression of fetal hemoglobin. This study contains RNA-seq and CUT&RUN data of a novel target, WIZ.
2024-06-19 | GSE247096 | GEO
Project description:The fetal (HbF)-to-adult (HbA) hemoglobin switch is a paradigm for developmental gene expression control with relevance to sickle cell disease and beta-thalassemia. Polycomb repressive complex (PRC) proteins regulate this switch, and an inhibitor of the EED subunit of PRC2 entered clinical trials for fetal hemoglobin activation. Yet, how PcG complexes function in this process, their target genes and relevant subunit composition are unknown. Here, we identified the PRC1 subunit BMI1 as a novel HbF repressor. We uncovered LIN28B, IGF2BP1, and IGF2BP3 as direct BMI1 targets and demonstrate that they account for the BMI1 effects. BMI1 functions as part of the canonical PRC1 (cPRC1) subcomplex as revealed by the physical and functional dissection of BMI1 protein partners. Lastly, we demonstrate that BMI1/cPRC1 acts in concert with the PRC2 to repress HbF through the same target genes. Our study illuminates how PRC silences HbF, highlighting an epigenetic mechanism involved in hemoglobin switching.
2023-03-01 | GSE218232 | GEO