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

Abstract Global warming has dominated worldwide climate change trends, and adaptability to high temperatures is the main factor underlying the spread of the pest Calliptamus italicus in Xinjiang Province, China. However, knowledge about the molecular mechanisms responsible for this adaptability and other related biological properties of C. italicus remain relatively unclear. Real-time quantitative polymerase chain reaction (RT-qPCR) is a key tool for gene expression analysis associated with various biological processes. Reference genes are necessary for normalizing gene expression levels across samples taken from specific experimental conditions. In this study, transcript level of five genes (GAPDH, 18S, TUB, ACT, and EF1α), commonly used as reference genes, were evaluated under nine different temperatures (27, 30, 33, 36, 39, 42, 45, 48, and 51°C) to assess their expression stability and further select the most suitable to be used on normalization of target gene expression data. Gene expression profiles were analyzed using geNorm, NormFinder, and BestKeeper software packages. The combined results demonstrated that the best-ranked reference genes for C. italicus are EF1α, GAPDH, and ACT under different thermal stress conditions. This is the first study that assesses gene expression analysis across a range of temperatures to select the most appropriate reference genes for RT-qPCR data normalization in C. italicus. These results should assist target gene expression analysis associated with heat stress in C. italicus.

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QRT-PCR Gene Expression Analysis in Monocytes from Normal and Alpha 1-Antitrypsin Deficient Individuals Using Total RNA or SideStep Cell Lysates.

10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a1895 ◽

2009 ◽

Author(s):

N Novoradovskaya ◽

CS Sandstrom ◽

LS Basehore ◽

T Sveger ◽

S Janciauskiene ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Total Rna ◽

Cell Lysates ◽

Qrt Pcr ◽

Alpha 1 Antitrypsin

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

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e23089 ◽

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]

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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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TWO MODIFIED RNA EXTRACTION METHODS COMPATIBLE WITH TRANSCRIPT PROFILING AND GENE EXPRESSION ANALYSIS FOR COTTON ROOTS

Preparative Biochemistry & Biotechnology ◽

10.1080/10826068.2012.759967 ◽

2013 ◽

Vol 43 (5) ◽

pp. 500-511 ◽

Cited By ~ 4

Author(s):

Chengjian Xie ◽

Chunyan Wang ◽

Xiaokun Wang ◽

Xingyong Yang

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Rna Extraction ◽

Extraction Methods ◽

Transcript Profiling ◽

Rna Extraction Methods

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

Journal of the Serbian Chemical Society ◽

10.2298/jsc091223084j ◽

2010 ◽

Vol 75 (8) ◽

pp. 1053-1061 ◽

Cited By ~ 2

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.

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