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United States Department of Agriculture

Agricultural Research Service

Title: A Method for Constructing Internal Standards for Use in Competitive Pcr

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

Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 20, 1995
Publication Date: N/A

Interpretive Summary: Our work concentrates on the study of variations in host immune responses resulting from parasite invasion. The use of Competitive Polymerase Chain Reaction is rapidly becoming an important method for quantitating gene expression in such studies, primarily in research areas where sample size is limiting. However, the methodology used to generate external standards for competitive PCR is expensive, time consuming and cumbersome. To date, no simple techniques have been designed to circumvent the problems associated with current practices. Herein, we have developed a rapid and simplistic approach to the construction of PCR competitor molecules that can be accomplished in a single day and with a high degree of success. This method will have general applicability across all fields of research and will specifically expedite our work in identifying host immunological responses involved in attenuating parasitic infections.

Technical Abstract: The application of quantitative polymerase chain reaction (RT-PCR) has become an indispensable tool for studying low levels of gene expression. A simple and rapid technique has been developed for the generation of exogenous competitor molecules that can be utilized during competitive PCR. This technique, which requires a single PCR amplification and a previously cloned sequence, uses internal primers separated by a predefined distance and designed to amplify in opposite directions and thus encompass the entire vector during the amplification process. The resultant product is a competitor molecule containing a region of sequence deletion relative to the native cDNA which is smaller than the naturally-derived cDNA sequence. The development of this methodology has allowed quantification of bovine interleukin 10 gene expression from stimulated and non-stimulated bovine cell populations that results from parasite invasion, without the need for radioisotope labeling. This as well as other similarly derived assays will allow the delineation of the mechanisms by which cattle become immune to economically important parasites.

Last Modified: 9/20/2014