scholarly journals Gene Expression Profiling of Reticulocytes in Patients with Hereditary Spherocytosis after Depleting Globin Gene Transcripts

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1255-1255
Author(s):  
Reena Das ◽  
Aggarwal Anu ◽  
Manu Jamwal ◽  
Prashant Sharma ◽  
Man Updesh Singh Sachdeva ◽  
...  

Abstract Introduction Global transcriptome analysis of circulating reticulocytes using microarrays is challenging due to the high abundance of globin transcripts. In order to accurately measure the transcriptome in reticulocytes, we planned to study the reticulocytes transcriptome profile before and after globin depletion. Patients with Hereditary Spherocytosis (HS) were recruited because of high reticulocytosis and also there was no global trancriptome profile available for the disease to the best of our knowledge. Methods Reticulocytes were purified by passing EDTA anticoagulated red cell suspensions through a column of microcrystalline cellulose and α-cellulose mixture. The extraction of total RNA was done fresh prior to microarray analysis using TRIzol reagent (Invitrogen) and RNeasy columns as per manufacturer's instruction. The globin transcripts were depleted with starting quantity of 3 µg of total RNA.using GLOBINclearTMHuman kit (Ambion, Austin, TX). RNA quality were assessed before and after depletion using Agilent Technologies 2100 Bioanalyzer in each sample used for the study. The Agilent Low Input Quick Amp Labelling kit was used to generate cRNA with a sample input of 200 ng total RNA in single-color microarray analysis (SurePrint G3 Human Gene Expression v3 8x60K Microarray, G4858A- 072363). cRNA was hybridized for 17 hours at 65˚C. After thorough washing, the results were extracted with Feature Extraction Project software and analysed using GeneSpring v13.0. For this study, differentially expressed genes were identified according to the criteria: t-test unpaired p (Corr) cut-off = 0.05 and fold-change cut-off of 2.0. These differentially expressed genes were imported in PANTHER (Protein Analysis Through Evolutionary Relationships) classification system to classify the genes. Results Transcriptome profiling of reticulocyte RNA from HS patients revealed 5318 annotated transcripts and 2744 lnc-RNA/uncharacterized transcripts to be differentially regulated before globin depletion. After globin depletion increase (8196 annotated transcripts and 4292 lnc-RNA) in the number of differentially regulated genes were observed. An increase of 54% and 56% was seen in annotated transcripts and noncoding transcripts respectively after globin depletion. This increased coverage may be attributed to the removal of the globin transcript. Gene Ontology analysis for molecular function, biological process, cellular component and protein class did not reveal any difference in the percentage of the category, though the number of transcripts was more after depletion. There were approximately 7% more pathways in the globin transcript depleted samples. Namely, 2-arachidonoylglycerol biosynthesis, biotin biosynthesis, carnitine metabolism, cobalamin biosynthesis, coenzyme a linked carnitine metabolism, cysteine biosynthesis, flavin biosynthesis, phenylalanine biosynthesis, sulfate assimilation, tyrosine biosynthesis pathways, etc. were elucidated only after globin depletion. Conclusions The globin transcript reduction followed by microarray analysis represents a robust methodology for studying pathophysiology of hematologic diseases which are not related to globin chain disorders. Furthermore we describe the global transcriptome profile of HS for the first time. Globin transcript depletion elucidates the better number of the transcripts and eventually the pathways which may have been earlier masked under the abundance of globin mRNAs. Pathway analysis and validation experiments are required to explain the role of these differentially expressed genes in the pathophysiology of HS. Disclosures No relevant conflicts of interest to declare.

2019 ◽  
Author(s):  
Jiasheng Xu ◽  
Kaili Liao ◽  
ZHONGHUA FU ◽  
ZHENFANG XIONG

Abstract Objective: To screen and analyze differentially expressed genes in pancreatic carcinoma tissues taken from Mongolian and Han patients by Affymetrix Genechip. Methods: Pancreatic ductal cell carcinoma tissues were collected from the Mongolian and Han patients undergoing resection in the Second Affiliated Hospital of Nanchang University from March 2015 to May 2018 and the total RNA was extracted. Differentially expressed genes were selected from the total RNA qualified by Nanodrop 2000 and Agilent 2100 using Affymetrix and a cartogram was drawn; The gene ontology (GO) analysis and Pathway analysis were used for the collection and analysis of biological information of these differentially expressed genes. Finally, some differentially expressed genes were verified by real-time PCR. Results: Through the microarray analysis of gene expression, 970 differentially expressed genes were detected by comparing pancreatic cancer tissue samples between Mongolian and Han patients. A total of 257 genes were significantly up-regulated in pancreatic cancer tissue samples in Mongolian patients; while a total of 713 genes were down-regulated. In the Gene Ontology database, 815 differentially expressed genes were identified with clear GO classification, and CPB1 gene showed the highest increase in expression level (multiple difference: 31.76). The pathway analysis detected 28 signaling pathways that included these differentially expressed genes, involving a total of 178 genes. Among these pathways, the enrichment of differentially expressed genes in the FAK signaling pathway was the strongest and COL11A1 gene showed the highest multiple difference (multiple difference: 5.02). The expression of differentially expressed genes CPB1, COL11A1、ITGA4、BIRC3、PAK4、CPA1、CLPS、PIK3CG and HLA-DPA1 determined by real-time PCR were consistent with the results of gene microarray analysis. Conclusions: The results of microarray analysis of gene expression profiles showed that there are a large number of differentially expressed genes in pancreatic cancer tissue samples comparing Mongolian and Han population. These genes are closely related to the cell proliferation, differentiation, invasion, metastasis and multi-drug resistance in pancreatic cancer. They are also involved in the regulation of multiple important signaling pathways in organisms.


2020 ◽  
Author(s):  
Jiasheng Xu ◽  
Kaili Liao ◽  
Zhonghua Fu ◽  
Zhenfang Xiong

Abstract Objective: To screen and analyze differentially expressed genes in pancreatic carcinoma tissues taken from Mongolian and Han patients by Affymetrix Genechip. Methods: Pancreatic ductal cell carcinoma tissues were collected from the Mongolian and Han patients undergoing resection in the Second Affiliated Hospital of Nanchang University from March 2015 to May 2018 and the total RNA was extracted. Differentially expressed genes were selected from the total RNA qualified by Nanodrop 2000 and Agilent 2100 using Affymetrix and a cartogram was drawn; The gene ontology (GO) analysis and Pathway analysis were used for the collection and analysis of biological information of these differentially expressed genes. Finally, some differentially expressed genes were verified by real-time PCR. Results: Through the microarray analysis of gene expression, 970 differentially expressed genes were detected by comparing pancreatic cancer tissue samples between Mongolian and Han patients. A total of 257 genes were significantly up-regulated in pancreatic cancer tissue samples in Mongolian patients; while a total of 713 genes were down-regulated. In the Gene Ontology database, 815 differentially expressed genes were identified with clear GO classification, and CPB1 gene showed the highest increase in expression level (multiple difference: 31.76). The pathway analysis detected 28 signaling pathways that included these differentially expressed genes, involving a total of 178 genes. Among these pathways, the enrichment of differentially expressed genes in the FAK signaling pathway was the strongest and COL11A1 gene showed the highest multiple difference (multiple difference: 5.02). The expression of differentially expressed genes CPB1, COL11A1、ITGA4、BIRC3、PAK4、CPA1、CLPS、PIK3CG and HLA-DPA1 determined by real-time PCR were consistent with the results of gene microarray analysis. Conclusions: The results of microarray analysis of gene expression profiles showed that there are a large number of differentially expressed genes in pancreatic cancer tissue samples comparing Mongolian and Han population. These genes are closely related to the cell proliferation, differentiation, invasion, metastasis and multi-drug resistance in pancreatic cancer. They are also involved in the regulation of multiple important signaling pathways in organisms.


2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Yue Lu ◽  
Yao Qi ◽  
Li Li ◽  
Yuhong Yan ◽  
Danni Yao ◽  
...  

Background. This study aimed to explore the mechanisms of action of the PSORI-CM01 and Yinxieling formulas in the treatment of patients with psoriasis vulgaris by analyzing gene expression in peripheral blood mononuclear cells (PBMCs). Methods. PBMC samples were collected from 21 patients before and after treatment. The study included nine patients in the PSORI-CM01 treatment group, 12 patients in the Yinxieling treatment group, and nine patients in the healthy control group. Gene expression levels in PBMCs were determined using the Affymetrix gene chip technology. Results. In the PSORI-CM01 group before and after treatment, a total of 668 differentially expressed genes were found, of which 445 were upregulated and 223 were downregulated. Before and after Yinxieling treatment, 657 differentially expressed genes were found, of which 168 were upregulated and 489 were downregulated. Venn analysis showed that 78 genes were not differentially expressed in the PSORI-CM01 group and 74 were not differentially expressed in the Yinxieling group compared with those in the controls. Among these genes, 72 genes were common to both groups, which were the genes on which the two drugs acted jointly. The results of KEGG analysis and Venn analysis on the signalling pathways of drug action in treatment groups showed that haemostasis and pathways involving Rho GTPases were common signalling pathways of drug action in the two groups. Conclusions. By a comparative analysis of the treatment groups, we found that both drugs have a positive effect on patients with psoriasis vulgaris, primarily by regulating the pathways related to platelet activation, aggregation, and blood coagulation. Trial registration: ChiCTR, ChiCTR-TRC-14005185, Registered 8 August 2014, http://www.chictr.org.cn/showproj.aspx?proj=4390


2020 ◽  
Author(s):  
Jiasheng Xu ◽  
Kaili Liao ◽  
ZHONGHUA FU ◽  
ZHENFANG XIONG

Abstract Objective: To screen and analyze differentially expressed genes in pancreatic carcinoma tissues taken from Mongolian and Han patients by Affymetrix Genechip. Methods: Pancreatic ductal cell carcinoma tissues were collected from the Mongolian and Han patients undergoing resection in the Second Affiliated Hospital of Nanchang University from March 2015 to May 2018 and the total RNA was extracted. Differentially expressed genes were selected from the total RNA qualified by Nanodrop 2000 and Agilent 2100 using Affymetrix and a cartogram was drawn; The gene ontology (GO) analysis and Pathway analysis were used for the collection and analysis of biological information of these differentially expressed genes. Finally, some differentially expressed genes were verified by real-time PCR. Results: Through the microarray analysis of gene expression, 970 differentially expressed genes were detected by comparing pancreatic cancer tissue samples between Mongolian and Han patients. A total of 257 genes were significantly up-regulated in pancreatic cancer tissue samples in Mongolian patients; while a total of 713 genes were down-regulated. In the Gene Ontology database, 815 differentially expressed genes were identified with clear GO classification, and CPB1 gene showed the highest increase in expression level (multiple difference: 31.76). The pathway analysis detected 28 signaling pathways that included these differentially expressed genes, involving a total of 178 genes. Among these pathways, the enrichment of differentially expressed genes in the FAK signaling pathway was the strongest and COL11A1 gene showed the highest multiple difference (multiple difference: 5.02). The expression of differentially expressed genes CPB1, COL11A1、ITGA4、BIRC3、PAK4、CPA1、CLPS、PIK3CG and HLA-DPA1 determined by real-time PCR were consistent with the results of gene microarray analysis. Conclusions: The results of microarray analysis of gene expression profiles showed that there are a large number of differentially expressed genes in pancreatic cancer tissue samples comparing Mongolian and Han population. These genes are closely related to the cell proliferation, differentiation, invasion, metastasis and multi-drug resistance in pancreatic cancer. They are also involved in the regulation of multiple important signaling pathways in organisms.


2020 ◽  
Vol 21 (21) ◽  
pp. 7924
Author(s):  
Motaz M. Fadul ◽  
Paul R. Heath ◽  
Johnathan Cooper-Knock ◽  
Julian M. Kurz ◽  
Hayder A. Al-Azzawi ◽  
...  

White matter lesions (WML) are a common feature of the ageing brain associated with cognitive impairment. The gene expression profiles of periventricular lesions (PVL, n = 7) and radiologically-normal-appearing (control) periventricular white matter cases (n = 11) obtained from the Cognitive Function and Ageing Study (CFAS) neuropathology cohort were interrogated using microarray analysis and NanoString to identify novel mechanisms potentially underlying their formation. Histological characterisation of control white matter cases identified a subgroup (n = 4) which contained high levels of MHC-II immunoreactive microglia, and were classified as “pre-lesional.” Microarray analysis identified 2256 significantly differentially-expressed genes (p ≤ 0.05, FC ≥ 1.2) in PVL compared to non-lesional control white matter (1378 upregulated and 878 downregulated); 2649 significantly differentially-expressed genes in “pre-lesional” cases compared to PVL (1390 upregulated and 1259 downregulated); and 2398 significantly differentially-expressed genes in “pre-lesional” versus non-lesional control cases (1527 upregulated and 871 downregulated). Whilst histological evaluation of a single marker (MHC-II) implicates immune-activated microglia in lesion pathology, transcriptomic analysis indicates significant downregulation of a number of activated microglial markers and suggests established PVL are part of a continuous spectrum of white matter injury. The gene expression profile of “pre-lesional” periventricular white matter suggests upregulation of several signalling pathways may be a neuroprotective response to prevent the pathogenesis of PVL.


2012 ◽  
Vol 44 (22) ◽  
pp. 1098-1106 ◽  
Author(s):  
Mohamed T. Ghorbel ◽  
Amir Mokhtari ◽  
Maimuna Sheikh ◽  
Gianni D. Angelini ◽  
Massimo Caputo

In cyanotic patients undergoing repair of heart defects, high level of oxygen during cardiopulmonary bypass (CPB) leads to greater susceptibility to myocardial ischemia and reoxygenation injury. This study investigates the effects of controlled reoxygenation CPB on gene expression changes in cyanotic hearts of patients undergoing surgical correction of tetralogy of Fallot (TOF). We randomized 49 cyanotic TOF patients undergoing corrective cardiac surgery to receive either controlled reoxygenation or hyperoxic/standard CPB. Ventricular myocardium biopsies were obtained immediately after starting and before discontinuing CPB. Microarray analyses were performed on samples, and array results validated with real-time PCR. Gene expression profiles before and after hyperoxic/standard CPB revealed 35 differentially expressed genes with three upregulated and 32 downregulated. Upregulated genes included two E3 Ubiquitin ligases. The products of downregulated genes included intracellular signaling kinases, metabolic process proteins, and transport factors. In contrast, gene expression profiles before and after controlled reoxygenation CPB revealed only 11 differentially expressed genes with 10 upregulated including extracellular matrix proteins, transport factors, and one downregulated. The comparison of gene expression following hyperoxic/standard vs. controlled reoxygenation CPB revealed 59 differentially expressed genes, with six upregulated and 53 downregulated. Upregulated genes included PDE1A, MOSC1, and CRIP3. Downregulated genes functionally clustered into four major classes: extracellular matrix/cell adhesion, transcription, transport, and cellular metabolic process. This study provides direct evidence that hyperoxic CPB decreases the adaptation and remodeling capacity in cyanotic patients undergoing TOF repair. This simple CPB strategy of controlled reoxygenation reduced the number of genes whose expression was altered following hyperoxic/standard CPB.


2011 ◽  
Vol 16 (6) ◽  
pp. 655-667 ◽  
Author(s):  
An Lu ◽  
Huichuan Wang ◽  
Xiaolin Hou ◽  
Huanrong Li ◽  
Guilin Cheng ◽  
...  

Ambient temperature is a critical factor that affects biological organisms in many ways. In this study, the authors investigated gene expression changes in rat small intestine in response to heat stress. Male Sprague-Dawley rats were randomly divided into control and heat-stressed groups. Both groups were housed at 25 °C, although the heat-stressed group was also subjected to 40 °C for 2 h each day for 10 successive days. Rats were sacrificed 1, 3, 6, and 10 days after heat treatment, and sections of their small intestine epithelial tissue were excised for morphological examination and microarray analyses. The rat rectal and body surface temperatures and serum cortisol levels were all significantly increased after heat treatment (p < 0.05). The jejuna were significantly damaged by 3 days after heat treatment began. Microarray analysis showed that 422 genes were differentially expressed, of which 290 genes were significantly upregulated and 132 genes were significantly downregulated. Subsequent bioinformatics analyses revealed that the differentially expressed genes were mainly related to stress, immune regulation, and metabolism processes. The bioinformatics analysis of the differentially expressed genes should be beneficial to further investigations on the underlying mechanisms involved in heat stress–induced damage in the small intestine.


2016 ◽  
Vol 8 (1) ◽  
pp. 17-32
Author(s):  
Marjorie Maharaj ◽  

You are here: Home › Differential Gene Expression after Emotional Freedom Techniques (EFT) Treatment: A Novel Pilot Protocol for Salivary mRNA Assessment doi 10.9769/EPJ.2016.8.1.MM Marjorie E. Maharaj, Department of Applied Psychology, Akamai University, Hilo, HI Abstract: Biopsychology is a rapidly expanding field of study since the completion of the Human Genome Project in 2003. There is little data measuring the effect of psychotherapeutic interventions on gene expression, due to the technical, logistical, and financial requirements of analysis. Being able to measure easily the effects of therapeutic experiences can validate the benefits of intervention. In order to test the feasibility of gene expression testing in a private practice setting, this study compared messenger ribonucleic acid (mRNA) and gene expression before and after psychotherapy and a control condition. With four non-clinical adult participants, it piloted a novel methodology using saliva stored at room temperature. A preliminary test of the interleukin- 8 (IL8) gene in both blood and saliva was performed in order to determine equivalency in the two biofluids; convergent validity was found. Following saliva test validation, a broad, genome-wide analysis was performed to detect differential gene expression in samples collected before and after treatment with Emotional Freedom Techniques (EFT), an evidence-based practice combining acupressure and cognitive exposure. The control treatment was non-therapeutic social interaction. To establish a baseline, participants received the control first, followed a week later by EFT. Analysis of samples was performed at three time points: immediately before treatment, immediately after, and 24 hours later. Differential expression between EFT and control was found in numerous genes implicated in overall health (p < 0.05). Further, the differentially expressed genes in this study were shown to be linked to immunity, pro or anti-inflammatory, as well as neuronal processes in the brain. Ten of the 72 differentially expressed genes are identified as promising targets for downstream research. The data show promise for the future use of salivary samples to determine the effects of therapy; this pilot protocol also illustrated the challenges and limitations of novel technologies employed in biopsychology. Keywords: epigenetics, DNA, mRNA, gene expression, protein synthesis, brain plasticity, neurogenesis, biopsychology


2019 ◽  
Author(s):  
Jiasheng Xu ◽  
Kaili Liao ◽  
ZHONGHUA FU ◽  
ZHENFANG XIONG

Abstract Objective: To screen and analyze differentially expressed genes in pancreatic carcinoma tissues taken from Mongolian and Han patients by Affymetrix Genechip. Methods: Pancreatic ductal cell carcinoma tissues were collected from the Mongolian and Han patients undergoing resection in the Second Affiliated Hospital of Nanchang University from March 2015 to May 2018 and the total RNA was extracted. Differentially expressed genes were selected from the total RNA qualified by Nanodrop 2000 and Agilent 2100 using Affymetrix and a cartogram was drawn; The gene ontology (GO) analysis and Pathway analysis were used for the collection and analysis of biological information of these differentially expressed genes. Finally, some differentially expressed genes were verified by real-time PCR. Results: Through the microarray analysis of gene expression, 970 differentially expressed genes were detected by comparing pancreatic cancer tissue samples between Mongolian and Han patients. A total of 257 genes were significantly up-regulated in pancreatic cancer tissue samples in Mongolian patients; while a total of 713 genes were down-regulated. In the Gene Ontology database, 815 differentially expressed genes were identified with clear GO classification, and CPB1 gene showed the highest increase in expression level (multiple difference: 31.76). The pathway analysis detected 28 signaling pathways that included these differentially expressed genes, involving a total of 178 genes. Among these pathways, the enrichment of differentially expressed genes in the FAK signaling pathway was the strongest and COL11A1 gene showed the highest multiple difference (multiple difference: 5.02). The expression of differentially expressed genes CPB1, COL11A1、ITGA4、BIRC3、PAK4、CPA1、CLPS、PIK3CG and HLA-DPA1 determined by real-time PCR were consistent with the results of gene microarray analysis. Conclusions: The results of microarray analysis of gene expression profiles showed that there are a large number of differentially expressed genes in pancreatic cancer tissue samples comparing Mongolian and Han population. These genes are closely related to the cell proliferation, differentiation, invasion, metastasis and multi-drug resistance in pancreatic cancer. They are also involved in the regulation of multiple important signaling pathways in organisms.


2017 ◽  
Vol 29 (1) ◽  
pp. 124
Author(s):  
T. J. Sood ◽  
S. Viviyan ◽  
S. K. Singla ◽  
M. Mukesh ◽  
M. S. Chauhan ◽  
...  

Although the blastocyst rate obtained with nuclear transferred (NT) embryos is higher than that obtained following in vitro fertilization (IVF) in buffalo, the live birth rate of NT embryos is <2% across different farm animal species compared with a birth rate >40% obtained with IVF embryos. This is believed to be due primarily to incomplete or incorrect nuclear reprogramming of the donor somatic cell by the oocyte, which results in aberrant embryonic gene expression. We compared the global transcriptome profile of buffalo blastocysts produced by hand-made cloning (HMC) and IVF using next-generation sequencing (NGS) for discovering transcripts that are differentially expressed between the 2 types of embryos. NT blastocysts were produced using fibroblast donor cells obtained from ear skin of a buffalo bull. The semen of the same bull was used for producing genetically half-identical IVF blastocysts. Total RNA was isolated from 3 pools of Day 8 NT and IVF blastocysts, with each pool containing 40 blastocysts. Complementary DNA library was prepared and subjected to NGS on Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA). The reads generated were aligned to Bos taurus reference genome, UMD 3.1. Differential expression analysis between the 2 blastocysts types at a minimum of 2-fold change revealed that 5557 transcripts were differentially expressed, of which 584 were unique to NT blastocysts, 709 were unique to IVF blastocysts, and 4264 were expressed in both types of blastocysts. Among these transcripts, at a significance level of P < 0.05, 331 transcripts were differentially expressed between the 2 blastocyst types, of which 19 were unique, 188 were down-regulated, and 143 were up-regulated in NT blastocysts. One-way ANOVA with Benjamini and Hochberg false discovery rate (FDR) correction was applied to determine the statistically significant differentially expressed transcripts. Nine of the differentially expressed transcripts (at minimal 2-fold change, P < 0.05), from different functional classes (RELN, NDRG1, SULT1A1, MAP1LC3A, MTHFD1L, PCBD1, PPA2, MGST1 and PRPH) were subjected to quantitative real-time PCR analysis for validation of NGS data. Gene expression level of RELN, NDRG1, SULT1A1, MAP1LC3A, PPA2, MGST1, and PRPH was found to be up-regulated while that of MTHFD1L and PCBD1 was down-regulated (P < 0.05) in NT embryos compared with IVF embryos. This pattern and the magnitude of relative gene expression level were found to be similar to that observed in NGS. These results indicate that the gene expression profile of NT embryos is very different from that of their IVF counterparts. Further analysis of these differentially expressed transcripts can help in identification of gene functional classes and pathways that are affected by the inefficient reprogramming of donor nuclei in NT embryos. Normalizing the expression of some of the differentially expressed genes may help in improving the cloning efficiency.


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