( 5C 3 min Step 3: 95C 30 sec ...)Simple Approaches for Optimization of RT-PCR

Step 4: 55C, 30 sec (or choose temperature based on primers)

Step 5: 72C, 1 min (or 1 to 2 min/kb)

Step 6: Go to step 3, 39 more times

Step 7: 72C, 3 min

6. ReactionStart thermal cycler program. When thermal cycler block reaches 42C, transfer reaction tube from ice to block. Upon completion of thermal cycler program, transfer tubes to ice.

7. AnalysisAnalyze sample (typically 1 to 10 μl aliquots) by agarose gel electrophoresis with staining by ethidium bromide. Visualize PCR product in gel with a UV transilluminator or fluorescence imager.

Results and Discussion

Optimizing sensitivity

Optimal sensitivity may be achieved by designing RT-PCR primers to accomplish detection of a target of any length based on amplification of a short (~0.5 kb) fragment of the target. In general, for each ~0.5 kb increase in the length of the designed RT-PCR product, approximately 10 to 100-fold more RNA should be used in order to detect the target (Fig. 2). Thus, the use of primers designed to amplify short fragments results in lower requirements for input RNA and greater ease in detection of targets.

Sensitivity may also be improved by the use of relatively high amounts of primers in RT-PCR reactions. For relatively short (0.5 kb) RT-PCR products in particular, increasing the concentration of each primer from 0.2μM to 1.2μM can improve sensitivity approximately 10-fold (Fig. 3). Note that this approach tends not to work as well with relatively high amounts of RNA or relatively long (1.5 kb) RT-PCR products. In such cases relatively high primer concentrations may result in decreased specificity and/or yield (see for example Fig. 3, 1 ng RNA, 1.2μM each primer).