scholarly journals Effects of Storage, RNA Extraction, Genechip Type, and Donor Sex on Gene Expression Profiling of Human Whole Blood

2007 ◽  
Vol 53 (6) ◽  
pp. 1038-1045 ◽  
Author(s):  
Sung Jae Kim ◽  
David J Dix ◽  
Kary E Thompson ◽  
Rachel N Murrell ◽  
Judith E Schmid ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Whole Blood ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Extraction Technique ◽  
Interindividual Differences ◽  
Human Whole Blood ◽  
Rna Quality

Abstract Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear which procedures alter results. In addition, characterization of interindividual and sex-based variation in gene expression is needed to understand sources and extent of variability. Methods: Whole blood was obtained from adult male and female volunteers (n = 42) and stored at various temperatures for various lengths of time. RNA was isolated and RNA quality analyzed. Affymetrix GeneChips (n = 23) were used to characterize gene expression profiles (GEPs) and to determine the effects on GEP of storage conditions, extraction techniques, types of GeneChip, or donor sex. Hierarchical clustering and principal component analysis were used to assess interindividual differences. Regression analysis was used to assess the relative impact of the studied variables. Results: Storage of blood samples for >1 week at 4 °C diminished subsequent RNA quality. Interindividual GEP differences were seen, but larger effects were observed related to RNA extraction technique, GeneChip, and donor sex. The relative importance of the variables was as follows: storage < genechip < extraction technique < donor sex. Conclusion: Sample storage and extraction methods and interindividual differences, particularly donor sex, affect GEP of human whole blood.

scholarly journals Three Years Follow-Up Study Showed Prenatal Methamphetamine Exposure May Cause Chronic Abnormalities In Transcriptomic Pattern

2021 ◽  
Author(s):  
Arvin Haghighatfard ◽  
Soha Seifollahi ◽  
Pegah Rajabi ◽  
Niloofar Rahmani ◽  
Rojin Ghannadzadeh
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Rna Extraction ◽  
Childbearing Age ◽  
Methamphetamine Use ◽  
Methamphetamine Use Disorder

Abstract Background: The high rate of methamphetamine use disorder among young adults and women of childbearing age makes it imperative to clarify the long-term effects of Methamphetamine exposure on the offspring. Behavioral and cognitive problems had been reported in children with parental Methamphetamine exposure (PME). The present study aimed to assess the acute and chronic effects of PME in molecular regulations and gene expression profiles of children during their first years of life.Methods: All subjects were recruited before birth, and sampling was conducted from the first ten days of birth, twelve months, twenty months, and thirty-six months of age. Finally, 2658 children with PME and 3573 normal children had been finished the follow-up. RNA extraction was operated from blood samples and gene expression profiling was conducted by using the Affymetrix GeneChip Human Genome U133 plus 2.0 Array Platform. Gene expression data were confirmed by Real-time PCR. Results: Gene expression profiling during thirty-six months showed several constant mRNA level alterations in children with PME compared with normal. These genes are involved in several gene ontologies and pathways involved with the immune system, neuronal functions, and bioenergetic metabolism. It seems that Methamphetamine use disorder before and during the pregnancy period may affect the expression profile of children, and these changes could remain years after birth. Affected genes have some similarities with the gene expression patterns of addiction, psychiatric disorders, neurodevelopmental disabilities, and immune deficiencies. Conclusion: Findings may shed light on the molecular effects of prenatal methamphetamine exposure and may lead to new psychological and somatic caring protocols for these children based on their potential abnormalities.

Gene expression profiling in human whole blood samples after controlled testosterone application and exercise

2011 ◽  
Vol 3 (10) ◽  
pp. 652-660 ◽  
Author(s):  
Martin Schönfelder ◽  
Hande Hofmann ◽  
Patricia Anielski ◽  
Detlef Thieme ◽  
Renate Oberhoffer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Whole Blood ◽  
Expression Profiling ◽  
Blood Samples ◽  
Human Whole Blood ◽  
Whole Blood Samples

scholarly journals Gene expression profiling of human whole blood samples with the Illumina WG-DASL assay

BMC Genomics ◽  
2011 ◽  
Vol 12 (1) ◽  
Author(s):  
Mary E Winn ◽  
Marian Shaw ◽  
Craig April ◽  
Brandy Klotzle ◽  
Jian-Bing Fan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Whole Blood ◽  
Expression Profiling ◽  
Blood Samples ◽  
Human Whole Blood ◽  
Whole Blood Samples

Gene expression profiling of the effects of intravenous immunoglobulin in human whole blood

2006 ◽  
Vol 43 (7) ◽  
pp. 939-949 ◽  
Author(s):  
Marco Ghielmetti ◽  
Michel Bellis ◽  
Martin O. Spycher ◽  
Sylvia Miescher ◽  
Guy Vergères
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Intravenous Immunoglobulin ◽  
Whole Blood ◽  
Expression Profiling ◽  
Human Whole Blood

scholarly journals Prenatal Methamphetamine Exposure could Cause Chronic Abnormalities in Transcriptomic Pattern; an Extended Follow-up Cohort Study

2021 ◽  
Author(s):  
Arvin Haghighatfard ◽  
Soha Seifollahi ◽  
Pegah Rajabi ◽  
Niloofar Rahmani ◽  
Rojin Ghannad zadeh
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Rna Extraction ◽  
High Rate ◽  
Childbearing Age ◽  
Methamphetamine Abuse

Abstract BackgroundThe high rate of methamphetamine abuse among young adults and women of childbearing age makes it imperative to clarify the long-term effects of Methamphetamine exposure on the offspring. Behavioral and cognitive problems had reported in children with parental Methamphetamine exposure (PME). The present study aimed to assess the acute and chronic effects of PME in molecular regulations and gene expression profiles of children during their first years of life.ResultsAll subjects were recruited before birth, and sampling was conducted from the first ten days of birth, twelve months, twenty months and thirty-six months of age. Finally, 2658 children with PME and 3573 normal children had been finished the follow-up. RNA extraction was operated from blood samples and gene expression profiling was conducted by using the Affymetrix GeneChip Human Genome U133 plus 2.0 Array Platform. Gene expression data were confirmed by Real-time PCR. Gene expression profiling during thirty-six months showed several constant mRNA level alterations in children with PME compared with normal. These genes are involved in several gene ontology and pathways involved with the immune system, neuronal functions and bioenergetic metabolism. It seems that Methamphetamine abuse before and during the pregnancy period may affect the expression profile of children, and these changes could be remain years after birth. Affected genes have some similarities to the gene expression patterns of addiction, psychiatric disorders, neurodevelopmental disabilities and immune deficiencies. ConclusionFindings may shed light on the molecular effects of prenatal methamphetamine exposure and may lead to new psychological and somatic caring protocols for these children based on their potential abnormalities.

Pharmacogenomic analysis of needle biopsies obtained before preoperative docetaxel/capecitabine/FEC (TX/FEC) chemotherapy for breast cancer

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 10595-10595 ◽  
Author(s):  
F. A. Holmes ◽  
J. A. O’Shaughnessy ◽  
B. Hellerstedt ◽  
J. Pippen ◽  
S. Vukelja ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Tumor Biology ◽  
Pathologic Complete Response ◽  
Research Network ◽  
Community Practice ◽  
Oncology Research

10595 Background: Our goal was to evaluate the feasibility of obtaining fine needle biopsies, for pharmacogenomic analysis, in community based oncology practices and develop gene expression-based predictors of pathologic complete response (pCR) to preoperative sequential docetaxel/capecitabine and 5-fluorouracil, epirubicin, cyclophosphamide chemotherapy. Methods: One hundred seventy-five patients were accrued at 29 sites in the US Oncology Research network. FNA specimens were mailed to a central laboratory (MDACC) and gene expression profiling was performed on Affymetrix U133A chips. Results: RNA extraction was started on 140 specimens, 112 of these (80%) yielded ≥1 μg total RNA, 69 were hybridized and 65 (94%) gene expression profiles have passed quality control as of abstract submission date. The analysis plan is to develop a multigene predictor of pCR from the first 80 cases and test its performance independently in the remaining cases. Conclusions: Collection of mandatory research FNA biopsies for pharmacogenomic research is feasible in community practice. Approximately 80% of biopsies yield sufficient RNA for gene expression profiling. In 20% of patients, either technical factors, which can be addressed, or tumor biology (necrotic, rapidly growing tumors) were limiting. Supported by Roche Laboratories, Inc., Nutley, NJ; Pfizer, New York, NY; and Precision Therapeutics, Pittsburgh, PA. [Table: see text]

Automated Sorting of Monocytes from Whole Blood for Reproducible Gene Expression Profiling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3840-3840
Author(s):  
Carsten Poggel ◽  
Timo Adams ◽  
Sabine Martin ◽  
Carola Pickel ◽  
Nicole Prahl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Blood Cell ◽  
Gene Expression Profiling ◽  
Whole Blood ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Specific Gene

Abstract Microarray-based gene expression profiling has been used to develop clinically relevant molecular classifiers for many different diseases. Furthermore, it has been shown for various chronic diseases that specific gene expression patterns are reflected at the level of blood cells. However, blood is a complex tissue comprising numerous cell types. Therefore, the contribution of rare cell types to a whole blood expression profile might not be detected and a substantial proportion of what is usually reported as “up-regulation” or “down-regulation” might actually be the result of a shift in cell populations and not of a true regulatory process. In order to circumvent these problems, several techniques have been established to analyze purified subpopulations rather than whole blood samples. Previously, it has been shown, for example, that reproducible gene expression profiles can be generated by positive selection of blood cell subsets from PBMCs1. As the preparation of PBMCs by, for example, Ficoll is time-consuming, inconvenient, and not amenable to automation, we have set up a combined direct whole blood cell separation and gene expression profiling protocol. By using Whole Blood CD14 MicroBeads in combination with the autoMACS Pro™ Separator, the separation protocol generally allowed enrichment of monocytes from whole blood within 30 min with purities higher than 90%. In combination with the depletion of neutrophils, the major source of contaminating RNA, purities increased to over 95% for all tested blood donors. Monocytes included the CD14bright/CD16− as well as the CD14dim/CD16+ populations. To assess the reproducibility of gene expression profiles and the influence of several experimental parameters, monocytes were sorted from 5 ml whole blood. RNA was extracted and hybridized to microarrays and the Pearson correlation coefficients of pairwise comparisons were calculated. Technical repeats of monocyte analysis from blood donated at different days showed a higher correlation coefficient than whole blood RNA. Blood storage at room temperature resulted in a strong deregulation of many genes, whereas blood stored at 4°C showed minimal changes, which is in agreement with previous studies. Skipping the centrifugation step, which is used to remove unbound MicroBeads did not alter the gene expression profiles. Incubation of sorted cells in PrepProtect™ Stabilization Buffer showed no alteration of gene expression thus enabling the shipping of cells without liquid nitrogen. Monocytes play a crucial role in diseases like atherosclerosis. Our rapid and simple protocol for combined direct cell sorting from whole blood and gene expression profiling of monocytes might help to ease the discovery of new biomarkers and to screen and monitor patients. 1 Lyons et al., BMC Genomics (2007), 8:64.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals Effectiveness of Gene Expression Profiling for Response Prediction of Rectal Adenocarcinomas to Preoperative Chemoradiotherapy

2005 ◽  
Vol 23 (9) ◽  
pp. 1826-1838 ◽  
Author(s):  
B. Michael Ghadimi ◽  
Marian Grade ◽  
Michael J. Difilippantonio ◽  
Sudhir Varma ◽  
Richard Simon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Response Prediction ◽  
Preoperative Chemoradiotherapy ◽  
Response To Therapy ◽  
Phase Iii ◽  
Correct Prediction

Purpose There is a wide spectrum of tumor responsiveness of rectal adenocarcinomas to preoperative chemoradiotherapy ranging from complete response to complete resistance. This study aimed to investigate whether parallel gene expression profiling of the primary tumor can contribute to stratification of patients into groups of responders or nonresponders. Patients and Methods Pretherapeutic biopsies from 30 locally advanced rectal carcinomas were analyzed for gene expression signatures using microarrays. All patients were participants of a phase III clinical trial (CAO/ARO/AIO-94, German Rectal Cancer Trial) and were randomized to receive a preoperative combined-modality therapy including fluorouracil and radiation. Class comparison was used to identify a set of genes that were differentially expressed between responders and nonresponders as measured by T level downsizing and histopathologic tumor regression grading. Results In an initial set of 23 patients, responders and nonresponders showed significantly different expression levels for 54 genes (P < .001). The ability to predict response to therapy using gene expression profiles was rigorously evaluated using leave-one-out cross-validation. Tumor behavior was correctly predicted in 83% of patients (P = .02). Sensitivity (correct prediction of response) was 78%, and specificity (correct prediction of nonresponse) was 86%, with a positive and negative predictive value of 78% and 86%, respectively. Conclusion Our results suggest that pretherapeutic gene expression profiling may assist in response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. The implementation of gene expression profiles for treatment stratification and clinical management of cancer patients requires validation in large, independent studies, which are now warranted.

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