|Skinner, Daniel - Dan|
Submitted to: Electronic Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/18/2004
Publication Date: 4/15/2004
Citation: Baek, K.-H. and Skinner, D.Z. 2004. Quantitative real-time pcr method to detect changes in specific transcript and total RNA amounts. Electronic Journal of Biotechnology. ISSN0717-3458. Vol. 7:1:55-60. Interpretive Summary: Methods commonly used for measuring the amount of expression of a specific gene ignore changes in the amount of expression of other genes in the system. We devised a method using a commonly employed technique to measure expression levels of all genes in a given tissue sample. Application of this technique will allow scientists to measure the impact of various treatments on global gene expression levels within a tissue sample of interest. This is an invited manuscript providing a non technical version of previously published work.
Technical Abstract: Genes exist in cells as DNA and are expressed by first being transcribed into an RNA copy. Actively expressed genes may be represented by thousands of RNA copies in a cell at any given time. In tissue exposed to a given treatment, the level of expression of several genes may change significantly due to changes in RNA synthesis or degradation, possibly altering the total amount of RNA in the tissue. One method used in studies to determine the change in the expression level of a gene of interest is known as quantitative real-time PCR (qRT-PCR). This method relies on the polymerase chain reaction (PCR), a technique which results in the multiplication of a specific DNA sequence; samples with more copies of the gene result in more rapid amplification of that gene. The specificity of the process is determined by the use of "primers," short pieces of DNA matching the gene of interest. The qRT- PCR process begins with converting the RNA back into DNA copies, known as cDNA (complementary DNA) using a naturally-occurring enzyme that performs this function, then amplifying the cDNA. The equipment used to carry out qRT- PCR determines the number of copies as they are formed, resulting in accurate comparisons of the original copy numbers of the gene in the samples. Typically, this method is used to estimate the number of copies of a transcript of interest in an aliquot of total RNA extracted from tissue samples before and after treatment. This determination is influenced by the amount of total RNA in the tissue. For example, if total RNA increased 2-fold, and the occurrence of the target transcript increased 10-fold, estimating the number of transcript copies in an aliquot of RNA would indicate only a five-fold increase in the target transcript, while a 10-fold increase had actually occurred in each cell. Here, we suggest a simple method using qRT-PCR to estimate the relative proportions of total RNA and a specific RNA transcript in tissue samples.