Use of a Translationally Coupled Reporter Gene to Eliminate Nonsense Mutations in a Random Mutagenesis Study

✍ Scribed by Jingdi Wang; Ruiwu Chen; Douglas A. Julin

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 53 KB
• Volume: 273
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4236

✦ Synopsis

copies per cell) but not MRP1 mRNA. Numerous published studies that employ Western blots, flow cytometry, and IC 50 measurements confirm these findings in a relative, not directly quantitative manner. Our results are the first to provide more accurate quantitative data that can be expressed as mRNA copies per cell (15,16).

A major advantage to our technique is that we can accurately quantitate multiple RNA samples simultaneously. We typically use all 96 wells of the 96-well block in our PCR machine to analyze 96 different samples. All reactions are initiated at exactly the same time and at a high temperature that increases the specificity of the analysis and, we believe, increases the sensitivity and accuracy of our quantitation. In other protocols, different reactions most likely initiate at slightly different times and temperatures and this would be predicted to add variability to the reversetranscription step. While we could analyze greater than 96 reactions at one time, we do not because the longer times required for reaction setups might allow some RNA degradation. If we wish to analyze a larger number of samples in one day, we first initiate 96 reactions simultaneously and then separately initiate a second set of 96 reactions in a time-staggered fashion. To date, we have used our technique to successfully quantitate at least 10 different mRNAs present in a variety of different cells and tissues. Our technique is thus broadly applicable to the study of any mRNA of interest in any cell or tissue of interest.