scholarly journals Yielding quality viral RNA by using two different chemistries: a comparative performance study

BioTechniques ◽  
2021 ◽  
Author(s):  
Jyotsnamayee Sabat ◽  
Subhra Subhadra ◽  
Sonalika Rath ◽  
Lal Mohan Ho ◽  
Srikanta Kanungo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Extraction Process ◽  
Performance Study ◽  
Silica Membrane ◽  
Comparative Performance ◽  
Magnetic Bead Separation

Purity and integrity are two important criteria for any RNA extraction process to qualify the RNA for meaningful gene expression analysis. This study compares four commercially available RNA extraction kits using silica membrane and magnetic bead separation methods. The performance was evaluated in terms of both quantity (total RNA amount in μg/μl) and purity (260/280 ratio). The concentration and purity of each kit was significantly different from those of the others (p < 0.001). Although quantity obtained from Mag MAX is comparatively lower than QIAGEN, the quality is comparable as evident from real-time PCR performance. This study suggests that there are practical differences between these RNA extraction kits that should be taken into account while isolating RNA required for gene expression analysis.

A new method for gene expression analysis of pure lymphocytes recovered from heterogeneous fixed mouse tissue.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23089-e23089
Author(s):  
Jennifer Chow ◽  
Ana Paula Galvão Da Silva ◽  
Gianni Medoro ◽  
Nicolò Manaresi ◽  
Paul David Lira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Tissue Sample ◽  
Critical Role ◽  
Rna Extraction ◽  
Response To Treatment ◽  
Mouse Tissue ◽  
Fixed Tissue ◽  
Mouse Splenocytes

e23089 Background: Tumor infiltrating lymphocytes (TILs) are biomarkers that play a critical role in cancer diseases, including differential diagnosis, determination of prognosis, prediction of response to treatment, and evaluation of disease progression. Gene expression analysis in TILs derived from fresh tissue may not accurately depict the gene profile of the tissue microenvironment as it can change aggressively during lymphocyte isolation and RNA extraction. In addition, tissue sample size can limit the isolation of TILs with current technologies. In this study, we demonstrate the use of the DEPArray™platform to isolate pure populations of lymphocytes from a fixed mouse tissue for RNA analysi. Methods: Mouse splenocytes were activated in vitro with anti-CD3 and -CD28 for 72hs. Cells were harvested, fixed with 2% paraformaldehyde (PFA) for 20 min at RT, and stained for either CD4 or CD8 expression. Gene expression analysis of CD45, ADORA2A, GLS and GAPDH was performed in CD4+ and CD8+ DEPArray™sorted cells using the TaqMan PreAmp Cells-to-Ct kit. Results: The table below summarizes the Ct values for CD45, ADORA2A, GLS and GAPDH expression in 300 fixed unsorted control and DEPArray™sorted lymphocytes. Conclusions: We have demonstrated the feasibility of gene expression analysis on pure populations of CD4+ and CD8+ cells isolated from a fixed tissue using the DEPArray™ platform. The advantage of this approach is the DEPArray’s ability to identify and isolate subpopulations of cells from complex heterogeneous samples and/or specimens that are limited by size or content. This methodology will be applied for isolation of TILs in syngeneic and xenograft models of cancers for downstream RNA applications. [Table: see text]

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

2010 ◽  
Vol 75 (8) ◽  
pp. 1053-1061 ◽  
Author(s):  
Ksenija Jakovljevic ◽  
Milena Spasic ◽  
Emina Malisic ◽  
Jelena Dobricic ◽  
Ana Krivokuca ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Whole Blood ◽  
Gene Expression Analysis ◽  
Expression Patterns ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Specific Gene ◽  
Isolation Methods ◽  
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.

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

2004 ◽  
Vol 37 (9) ◽  
pp. 741-744 ◽  
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

scholarly journals Optimization of the PAXgene™ blood RNA extraction system for gene expression analysis of clinical samples

2005 ◽  
Vol 19 (5) ◽  
pp. 182-188 ◽  
Author(s):  
Viengthong Chai ◽  
Aikaterini Vassilakos ◽  
Yoon Lee ◽  
Jim A. Wright ◽  
Aiping H. Young
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Rna Extraction ◽  
Extraction System ◽  
Clinical Samples

scholarly journals Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

2018 ◽  
Vol 18 (1) ◽  
Author(s):  
Rikki A. M. Brown ◽  
Michael R. Epis ◽  
Jessica L. Horsham ◽  
Tasnuva D. Kabir ◽  
Kirsty L. Richardson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Target Gene ◽  
Rna Extraction ◽  
Total Rna ◽  
Target Gene Expression ◽  
Total Rna Extraction

scholarly journals Comparative gene expression analysis of Fas and related genes in Comparative gene expression analysis of Fas and related genes in preeclamptic and healthy women: A cross-sectional study

2020 ◽  
Author(s):  
Zaima Ali ◽  
Saba Khaliq ◽  
Saima Zaki ◽  
Hafiz Usman Ahmad ◽  
Khalid Pervaiz Lone
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Mononuclear Cells ◽  
Rna Extraction ◽  
Control Group ◽  
Hypertensive Disorder ◽  
Cross Sectional ◽  
Tnf Α

Background: Preeclampsia is a hypertensive disorder of pregnancy affecting about 2-10% pregnancies worldwide. mRNA expression of tumor necrosis factor alpha (TNF-α), Fas, and FasL have been reported to be altered in placental bed in preeclamptic pregnancies. We hypothesized that the expression of these genes is also altered in peripheral blood mononuclear cells (PBMCs) in preeclampsia. Objective: To compare the expression of Fas receptor and related genes in PBMCs of preeclamptic and normotensive pregnant women. Materials and Methods: A cross-sectional comparative study comprising of 18 cases and 18 controls was designed. 5 ml of venous blood was drawn and collected considering aseptic measures. Buffy coat was separated by centrifugation and stored at –20°C. Favor Prep total RNA Isolation Kit (Favorgen, Taiwan) was used for RNA extraction. The mRNA expression of TNF-α, Fas, and FasL was measured by real-time polymerase chain reaction in PBMCs in preeclamptic and normal pregnancies. Results: A significant increase in mRNA expression of TNF-α, Fas, and FasL (p ≤ 0.001) was observed in PBMCs of preeclamptic pregnancies compared to the control group (p ≤ 0.001). Moreover, a significant positive correlation was found between the TNF-α mRNA expression and Fas and FasL (p ≤ 0.001). Conclusion: The results lead to the conclusion that mRNA expression of TNF-α, Fas, and FasL in the maternal PBMCs is altered in preeclamptic pregnancies and might contribute to the pathogenesis of the disease. Key words: Preeclampsia, TNF-α, Fas, Apoptosis.

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