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United States Department of Agriculture

Agricultural Research Service

Title: Quantification of Bovine Cytokine Gene Expression with Rnase Protection Assay and Competitive Rt-Pcr

Authors
item Canals, Ana
item Zarlenga, Dante
item Gasbarre, Louis

Submitted to: Veterinary Immunology International Symposium
Publication Type: Abstract Only
Publication Acceptance Date: February 22, 1995
Publication Date: N/A

Technical Abstract: Studies aimed at defining the role of cytokines in the generation of protective immune responses to gastrointestinal nematodes of cattle have been hampered by the lack of either monospecific bioassays to measure secreted products or assays to quantitate bovien cytokine mRNA expression. To address this limitation, two assays were devised to detect cytokine gene etranscripts. Specific probes for bovine IL-2, IL-4, IL-10, IL-12 and gamma-interferon were developed and used in RNase protection assays (RPA). These assays were able to detect cytokine gene expression in stimulated PBMC and abomasal lymph node cell populations using as little as 5 ug of total RNA. Results from the RPA represent a quick and semi-quantitative method for assessing cytokine gene expression, ideal for studying large numbers of samples. For low level expression of cytokines (nonstimulated populations), or when small amounts of RNA are available, a competitive RT-PCR was subsequently developed. Competitor molecules for the selected cytokines were generated by a novel techniqu, internal primers separated by a predefined distance were designed to amplify previously cloned sequences in opposite directions. This amplification, which encompasses the vector as well as the clone, results in a cloned molecule that upon amplification generates a product smaller than the native cDNA molecule. Although more laborioius, RT-PCR is more sensitive and offers a quantitative assessment of cytokine message from stimulated and non-stimulated cell populations without the need of radioisotope labeling.

Last Modified: 8/27/2014
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