Root sampling and RNA extraction methods for field-based gene expression analysis of soybeans

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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Evaluation of RNA extraction methods in rice and their application in expression analysis of resistance genes against Magnaporthe oryzae

Biotechnology & Biotechnological Equipment ◽

10.1080/13102818.2016.1259015 ◽

2016 ◽

Vol 31 (1) ◽

pp. 75-84 ◽

Cited By ~ 1

Author(s):

Parisa Azizi ◽

Mohd Y. Rafii ◽

Maziah Mahmood ◽

Siti Nor Akmar Abdullah ◽

Mohamed Musa Hanafi ◽

...

Keyword(s):

Expression Analysis ◽

Resistance Genes ◽

Magnaporthe Oryzae ◽

Rna Extraction ◽

Extraction Methods ◽

Rna Extraction Methods

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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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Evaluation of four RNA extraction methods for gene expression analyses of Cryptosporidium parvum and Toxoplasma gondii oocysts

Journal of Microbiological Methods ◽

10.1016/j.mimet.2012.03.010 ◽

2012 ◽

Vol 89 (3) ◽

pp. 185-192 ◽

Cited By ~ 5

Author(s):

Mary J. See ◽

Sarah E. Staggs ◽

J.P. Dubey ◽

Eric N. Villegas

Keyword(s):

Gene Expression ◽

Toxoplasma Gondii ◽

Cryptosporidium Parvum ◽

Rna Extraction ◽

Extraction Methods ◽

Gene Expression Analyses ◽

Rna Extraction Methods

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The Successful Use of Archived Formalin-Fixed Paraffin-Embedded (FFPE) Material for Gene Expression Studies.

Blood ◽

10.1182/blood.v108.11.4637.4637 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4637-4637

Author(s):

Konstantinos Lilakos ◽

Maria K. Angelopoulou ◽

George Georgiou ◽

Vassilios Salpeas ◽

Penelope Korkolopoulou ◽

...

Keyword(s):

Gene Expression ◽

Real Time Pcr ◽

Rna Extraction ◽

Extraction Methods ◽

Ct Number ◽

Expression Studies ◽

Ffpe Tissues ◽

Ffpe Material ◽

Rna Extraction Methods ◽

Gene Expression Studies

Abstract BACKGROUND: FFPE tissues are widely used as a source of genomic DNA (gDNA), as well as for immunohistochemistry. RNA amplification and its usage for gene expression studies from archived FFPE material has been hampered due to RNA degradation. AIM: The description of a protocol for RNA extraction from archived FFPE tissues followed by reverse transcription (RT) and real time PCR and its implementation in the study of reference genes. METHODS: RNA was extracted from single 15μm sections from 18 samples 30 months old and 6 samples 10 years old from patients with diffuse large B-cell lymphoma (DLBCL). The FFPE RNA Extraction kit (Roche) was used, followed by DNA digestion and run on an Agilent2100 Bioanalyzer. RT was performed using the Transcriptor System (Roche) with modified temperatures followed by real time PCR for the c-abl and Survivin genes using the Universal Library probes and primers (Roche). In addition for the 10-year old samples, beta2 microglobulin (b2m) and G6PDH were also amplified. RESULTS: RNA that was yielded, was around 250 bp. c-abl was successfully amplified in all 18 samples up to 30 months old with a median cycle (Ct) number of 33.74. All 3 control genes were successfully amplified from the 10-year old samples with a median Ct number of 30.3 for G6PDH, 23.4 for b2m and 28.6 for c-abl. To further evaluate the amplification potency we successfully amplified Survivin transcripts with a median Ct number of 35.2. Survivin/abl ratio was calculated at a median of 0.5 for all DLBCL samples. gDNA interferenence was excluded, since no amplification signal was observed when gDNA was used as a template. The special RNA extraction methods, elevated temperatures for RT, as well as the use of the Universal Library probes, which are specifically designed to be short (8–9bp) and hybridize to a short amplicon (72bp for c-abl and 97bp for Survivin) were the major novelties of this method. Conclusions: Specialized RNA extraction methods and the use of Universal Library Probes can succesfully lead to several target gene amplification from archived FFPE material and potentially provide templates for gene expression studies.

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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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