![]() ![]() The method was first described by Gilliland (7) and Becker-Andre (8). It may be necessary to use several house-keeping genes for this approach.Īnother method utilizes an exogenous internal standard in competitive PCR, during which one set of primers is used to amplify both the target cDNA and another DNA fragment (the internal standard)-in essence the second DNA fragment competes with the target DNA for the same primers and thus acts as an internal standard. However, expression of a putative “stable” housekeeping gene ( GAPDH or b-actin ) can be actually varied as much as that of the target gene (5, 6). One method uses an endogenous internal standard in a multiplex PCR, in which two sets of primers are used in the same PCR reaction-one set specific to the target gene cDNA and the other set specific to a gene transcript invariant in the experiment, such as a “housekeeping” gene (3, 4). They all involve the use of an internal standard to compare the efficiency of the PCR in different reactions. Several methods have been described to address this problem. This obscures differences in levels of the target mRNA during amplification. ![]() However, quantitation of mRNA levels or changes in mRNA levels can be problematic due to the exponential nature of PCR, where small variations in amplification efficiency can lead to dramatic changes in product yields. The use of PCR to examine levels of gene transcripts, often referred to as reverse transcriptase PCR (RT-PCR), has become very popular because it is sensitive and rapid (1,2). ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |