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]

Cancer activation pathways of thymic epithelial tumors (TETs) by targeted gene expression analysis.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 8575-8575
Author(s):  
Jose Carlos Benitez ◽  
Bastien Job ◽  
Vincent Thomas de Montpreville ◽  
Ludovic Lacroix ◽  
Patrick Saulnier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inflammatory Response ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Anterior Mediastinum ◽  
Response To Treatment ◽  
Molecular Characteristics ◽  
Thymic Epithelial Tumors ◽  
Activation Pathways ◽  
Cluster 2

8575 Background: TETs are rare malignancies of the anterior mediastinum. Clinical behavior varies from mild thymoma (T) A to aggressive thymic carcinoma (TC). The biology of TETs is poorly understood and knowledge of the transcriptomic fingerprint of T and TC is limited. Additionally, up to 30% of patients (pts) will develop associated autoimmune disorders (AIDs). We aimed to characterize the main cancer activation pathways of the TET subgroups. Methods: We selected a representative balanced set of Ts and TCs to analyze 24 main cancer activation pathways using HTG Oncology biomarkers panel (2562 genes) by RNA sequencing. Tumor representative paraffin-embedded blocks were macrodissected for gene expression analysis. We analyzed epidemiologic, clinical and pathological characteristics of pts with TET’s and correlated with genes expression based on cancer Hallmarks. Results: From January 2010 to December 2019, 219 pts were included in the cohort. Molecular results of 194 pts were available. Median age at diagnosis was 56 (9-83) years. 54.1% were women. 65/194 (33.5%) reported AIDs. T B2 was the most frequent (n=41, 21.1%), followed by B1, AB, B3, TC and A. RNA expression analysis identified 2 main clusters, corresponding mainly to T (cluster 2) and TC (cluster 1) respectively (p<0.0001) (Table). Tumors of cluster 1 (TCs predominant) shown activated pathways (MYC [gene ratio= 0.5]; p<0.0001). In cluster 2 (T predominant), activated pathways differed among subgroups: B1 (E2F[0.5], G2M checkpoint [0.45]; p<0.001), B2 (E2F [>0.4]; p<0.0001). Routes were mostly suppressed: A (MYC [>0.5], E2F[>0.4], G2M checkpoint[>0.45], mitotic spindle[>0.35], MTOR [<0.35]; p<0.001), AB (INFα [>0.5], inflammatory response [>0.45], INTɣ [0.45], NFkb [>0.4], MTOR[>0.4]; p<0.001), B1 (EMT [0.6], angiogenesis [>0.5], INFα [0.5], homeostasis [0.5], NFkb [0.5], INTɣ [>0.45], myogenesis [<0.4]; p<0.0001), B2 (INFα [>0.65], INTɣ [>0.5], NFkb [0.5], EMT [0.45], inflammatory response [<0.4]; p< 0.0001), B3 (EMT [0.6], MYC [0.45]; p<0.001). Among pts reporting AIDs 61 and 4 were associated to cluster 2 and 1, respectively (p=0.017). Conclusions: We describe differential molecular characteristics among histological subgroups in 2 clusters. The analysis suggests new therapeutic venues. Additional analysis will be presented on outliers and response to treatment.[Table: see text]

scholarly journals Genome-Wide Gene Expression Analysis Reveals a Critical Role for CRYPTOCHROME1 in the Response of Arabidopsis to High Irradiance

2007 ◽  
Vol 144 (3) ◽  
pp. 1391-1406 ◽  
Author(s):  
Tatjana Kleine ◽  
Peter Kindgren ◽  
Catherine Benedict ◽  
Luke Hendrickson ◽  
Åsa Strand
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Critical Role ◽  
High Irradiance ◽  
Genome Wide ◽  
Genome Wide Gene Expression

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

A lab-on-a-chip system integrating tissue sample preparation and multiplex RT-qPCR for gene expression analysis in point-of-care hepatotoxicity assessment

Lab on a Chip ◽  
2015 ◽  
Vol 15 (20) ◽  
pp. 4032-4043 ◽  
Author(s):  
Geok Soon Lim ◽  
Joseph S. Chang ◽  
Zhang Lei ◽  
Ruige Wu ◽  
Zhiping Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sample Preparation ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Tissue Sample ◽  
Point Of Care ◽  
Lab On A Chip

In this study, we realize an integrated lab-on-a-chip system with “sample-in-answer-out” multiplex gene expression analysis capabilities for point-of-care hepatotoxicity assessment.

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