Development and Use of a Plasmid Encoding Green Fluorescent Protein in Multiple Antibiotic-Resistant Salmonella

Timothy S. Frana, Steve A. Carlson
2001 BioTechniques  
specific RT-PCR products were not generated (lanes 3 and 12). The same results were obtained for the other six mRNAs investigated using S2-A2, S3-A3, S4-A4, S5-A5, S6-A6, and S7-A7 primer pairs (data not shown). Thus, the results presented in this article show that, depending on the reaction conditions (i.e., enzyme concentration, temperature, and buffer composition), RTases from different manufacturers demonstrate different specificity and background activity. Without preliminary selection of
more » ... inary selection of adequate RT conditions, the ability of different RTases to initiate the backround cDNA synthesis can lead to misinterpretation of RT-PCR results using recombinant RNA as an internal standard. This can be the case if the sequence of standard and studied RNAs are different; the presence of the background synthesis initiation sites on the studied RNA but not on the standard RNA (or visa versa) will result in different cDNA yield on the two RNAs and, consequently, in misinterpretation of the data. If the efficiency of cDNA synthesis from exogenous primer is comparable with, or much lower than, the efficiency of background cDNA synthesis, the measurement error will be significant. To minimize the measurement error, it is necessary either to use an internal RNA standard fully identical to the studied RNA or to select appropriate RT reaction conditions.
doi:10.2144/01301bm03 pmid:11196315 fatcat:ks5vjqya4nbhhfrhfszovqfw3i