Comparison of phenol-based and alternative RNA isolation methods for gene expression analyses

The widespread use of gene expression analyses has been limited by the lack of a critical evaluation of the methods used to extract nucleic acids from human tissues. For evaluating gene expression patterns in whole blood or leukocytes, the method of RNA isolation needs to be considered as a critical variable in the design of the experiment. Quantitative real-time PCR (qPCR) is widely used for the quantification of gene expression in today?s clinical practice. Blood samples as a preferred RNA source for qPCR should be carefully handled and prepared to not inhibit gene expression analyses. The present study was designed to compare the frequently used guanidine thiocyanate-phenol-chloroformbased method (TRI Reagent?) with two alternative RNA isolation methods (6100 PrepStation and QIAamp?) from whole blood or leukocytes for the purpose of gene expression analysis in chronic myeloid leukemia (CML) patients. Based on the results of this study, for the best combination of yield and RNA extraction purity, taking into account the necessary amount of the clinical sample and performance time, the protocol using phenol-based TRI Reagent? for RNA extraction from leukocytes is suggested as the most suitable protocol for this specific gene expression analysis.

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Research note: methodology for high-quality RNA extraction from poultry whole blood for further gene expression analysis

British Poultry Science ◽

10.1080/00071668.2014.888397 ◽

2014 ◽

Vol 55 (2) ◽

pp. 194-196 ◽

Cited By ~ 3

Author(s):

J. L. Mewis ◽

X. Sun ◽

M. J. Zuidhof ◽

L. L. Guan

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Whole Blood ◽

Gene Expression Analysis ◽

Rna Extraction ◽

Research Note ◽

High Quality

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Sensitive and Quantitative Measurement of Gene Expression Directly from a Small Amount of Whole Blood

Clinical Chemistry ◽

10.1373/clinchem.2005.065078 ◽

2006 ◽

Vol 52 (7) ◽

pp. 1294-1302 ◽

Cited By ~ 30

Author(s):

Zhi Zheng ◽

Yuling Luo ◽

Gary K McMaster

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reverse Transcription ◽

Whole Blood ◽

Quantitative Measurement ◽

Gene Expression Analysis ◽

Rna Isolation ◽

Multiplex Assay ◽

Quality Data ◽

Branched Dna

Abstract Background: Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. Methods: We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. Results: The single- and multiplex assays could quantitatively measure as few as 6000 and 24 000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 μL of whole blood. Both formats had CVs <10% and dynamic ranges of 3–4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Conclusions: Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

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Optimizing RNA extraction yield from whole blood for microarray gene expression analysis

Clinical Biochemistry ◽

10.1016/j.clinbiochem.2004.03.013 ◽

2004 ◽

Vol 37 (9) ◽

pp. 741-744 ◽

Cited By ~ 25

Author(s):

Jian Wang ◽

John F. Robinson ◽

Hafiz M.R. Khan ◽

David E. Carter ◽

James McKinney ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Whole Blood ◽

Gene Expression Analysis ◽

Rna Extraction ◽

Extraction Yield ◽

Microarray Gene Expression ◽

Microarray Gene ◽

Microarray Gene Expression Analysis

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Genome-Wide Identification and Gene Expression Analysis of ABA Receptor Family Genes in Brassica juncea var. tumida

Genes ◽

10.3390/genes10060470 ◽

2019 ◽

Vol 10 (6) ◽

pp. 470 ◽

Cited By ~ 2

Author(s):

Cheng ◽

Zhong ◽

Cai ◽

Su ◽

Keyword(s):

Gene Expression ◽

Brassica Juncea ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Expression Patterns ◽

Plant Response ◽

Specific Gene ◽

Cis Elements ◽

Physiological Processes ◽

Aba Receptor

Abscisic acid (ABA) plays important roles in multiple physiological processes, such as plant response to stresses and plant development. The ABA receptors pyrabactin resistance (PYR)/ PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) play a crucial role in ABA perception and signaling. However, little is known about the details regarding PYL family genes in Brassica juncea var. tumida. Here, 25 PYL family genes were identified in B. juncea var. tumida genome, including BjuPYL3, BjuPYL4s, BjuPYL5s, BjuPYL6s, BjuPYL7s, BjuPYL8s, BjuPYL10s, BjuPYL11s, and BjuPYL13. The results of phylogenic analysis and gene structure showed that the PYL family genes performed similar gene characteristics. By analyzing cis-elements in the promoters of those BjuPYLs, several hormone and stress related cis-elements were found. The results of gene expression analysis showed that the ABA receptor homologous genes were induced by abiotic and biotic stress. The tissue-specific gene expression patterns of BjuPYLs also suggested those genes might regulate the stem swelling during plant growth. These findings indicate that BjuPYLs are involved in plant response to stresses and organ development. This study provides valuable information for further functional investigations of PYL family genes in B. juncea var. tumida.

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IL-6R Protective Variant rs7529229 Reduces Interleukin-6 Signaling and Contributes To A Decreased Ischemic Stroke Risk

10.21203/rs.3.rs-93821/v1 ◽

2020 ◽

Author(s):

Haihua Zhang ◽

Wenbo Zhao ◽

Bian Liu ◽

Tao Wang ◽

Zhifa Han ◽

...

Keyword(s):

Gene Expression ◽

Ischemic Stroke ◽

Expression Analysis ◽

Interleukin 6 ◽

Whole Blood ◽

Gene Expression Analysis ◽

Minor Allele ◽

Specific Gene ◽

Healthy Controls ◽

Increased Risk

Abstract Background Interleukin-6 (IL-6) signaling is associated with an increased risk of coronary artery disease (CAD) and ischemic stroke (IS). Growing evidence shows that the minor alleles of IL-6 receptor gene (IL-6R) variants rs2228145, rs7529229, and rs4129267 significantly increase soluble IL-6R levels and reduce CAD risk. However, the role of IL-6R variants in IS is largely unknown, prompting us to perform a comprehensive analysis. Methods In stage 1 of this study, we performed a meta-analysis of three genome-wide association study datasets from MEGASTROKE, UK Biobank, and the Million Veteran Program to evaluate the association of rs7529229 with IS. In stage 2, we conducted an expression quantitative trait loci analysis to examine the effects of rs7529229 on IL-6R expression in neuropathologically healthy individuals from the UK Brain Expression Consortium, GTEx project, and the eQTLGen Consortium. In stage 3, we used a tissue-specific gene expression analysis to evaluate differences in IL-6R expression across human tissues using gene expression data from GTEx. In stage 4, we conducted a case–control gene expression analysis to explore the differential expression of IL-6R in the whole blood of IS patients and healthy controls. Results We found that: (1) the rs7529229 minor allele significantly reduced the risk of developing IS (odds ratio=0.97, 95% confidence interval 0.95–0.99, P=2.30E-03); (2) the rs7529229 minor allele significantly reduced IL-6R expression in relevant tissues especially in blood vessels and whole blood; (3) IL-6R was mainly expressed in skeletal muscle and whole blood; and (4) IL-6R expression was significantly reduced in the whole blood of healthy controls compared with IS patients. Importantly, the biological senses in stages 1–4 were all convergent. Conclusions Taken together, our findings indicate that the rs7529229 minor allele decreases IL-6R expression in relevant tissues, diminishes IL-6 signaling, and eventually reduces the IS risk. Hence, IL-6R may be a potential therapeutic target for IS. Tocilizumab, a monoclonal antibody that blocks both membrane-bound and circulating IL-6R, might be effective in treating IS or lowering its risk of development, so warrants further testing in suitably powered randomized controlled trials.

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Microfluidic Leukocyte Isolation for Gene Expression Analysis in Critically Ill Hospitalized Patients

Clinical Chemistry ◽

10.1373/clinchem.2007.099150 ◽

2008 ◽

Vol 54 (5) ◽

pp. 891-900 ◽

Cited By ~ 23

Author(s):

Aman Russom ◽

Palaniappan Sethu ◽

Daniel Irimia ◽

Michael N Mindrinos ◽

Steve E Calvano ◽

...

Keyword(s):

Gene Expression ◽

Critically Ill ◽

Expression Analysis ◽

Clinical Setting ◽

Whole Blood ◽

Gene Expression Analysis ◽

Expression Patterns ◽

Hospitalized Patients ◽

Genome Wide ◽

Genome Wide Expression

Abstract Background: Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood. Methods: We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique. Results: When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals. Conclusions: The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting.

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