Reverse transcription-polymerase chain reaction (RT-PCR) converts and amplifies a single-stranded RNA template to yield abundant double-stranded DNA product(1,2,3). RT-PCR is a powerful tool for analysis of gene expression and for characterization of RNA splice variants. RT-PCR is generally carried out as either one-step RT-PCR, in which all reagents are combined in one tube and RT and PCR are conducted sequentially in this tube(1,3) or two-step RT-PCR, in which RT and PCR are conducted sequentially in separate tubes(2). The USB One-Step RT-PCR Kit (PN 78350), Two-Step RT-PCR Kit (PN 78355), and RT Script Kit (PN 78360) are based on M-MLV Reverse Transcriptase(4) and Taq DNA Polymerase(5). M-MLV Reverse Transcriptase is useful in RT-PCR due to its naturally low RNase H activity(2). The kits are designed for typical routine analyses of gene expression, based on amplification of relatively short (≤ 1.5 kb) RT-PCR products (Fig. 1).
Optimization of reaction parameters is a crucial aspect of RT-PCR. With new targets, optimization can determine the difference between failure and success in generating product. With familiar targets, optimization can minimize use of sample and reagents and maximize reproducibility and reliability of results. A small investment of effort in optimizing reactions can yield a large payoff in results, particularly if careful attention is also paid to fundamental issues such as primer design(6,7), RNA isolation(8,9), and use of RNase free reagents(10). This Tech Tip describes several simple approaches for optimizing sensitivity, specificity, and yield in one-step RT-PCR. The same approaches also apply in two-step RT-PCR.
Reactions (50 μl) were carried out with the USB One-Step RT-PCR Kit (PN 78350) according to the kit protocol, except as no ted. Reactions and primers are described in Table 1. A brief version of the protocol is described here. Note that the reaction master mix can be scaled-up for analysis of multiple samples. Reaction master mixes and assembled reactions should be mixed well by vortexing.
1. RNA/Primer MixIn first tube, combine RNA and oligonucleotide primers to 10 μl vol on ice.
RNA x μl (total RNA: 1 pg to 1 μg)
Forward Primer (10μM) 2 μl (0.4μM in 50 μl reaction)
Reverse Primer (10μM) 2 μl (0.4μM in 50 μl reaction)
Water (RNase-free) 6-x μl (to adjust volume to 10 μl)
Mix well and collect contents at bottom of tube.
2. Heat Treatment of RNA/Primer (optional)Heat RNA/primer mixture at 75C, 5 min, then cool immediately on ice for at least 5 minutes and store on ice. Note that this step may be omitted in some cases but may be necessary in others.
3. Reaction Master MixIn second tube, combine RT-PCR reagents to 40 μl on ice.
Water (RNase-free) 27 μl
RT-PCR Buffer (5X) 10 μl
PCR Nucleotide Mix (10mM) 1 μl
RNase Inhibitor (4 units/μl) 1 μl
RT-PCR Enzyme Mix (50X) 1 μl
Mix contents of tube by vortexing, collect contents at bottom of tube, and store on ice.
4. Complete Reaction MixCombine 10 μl RNA/primer and 40 μl reaction master mix in one tube on ice for complete 50 μl reaction. Mix contents of tube by vortexing, collect contents at bottom of tube, and store on ice.
5. Thermal CycleDesign a thermal cycler program appropriate for RT-PCR amplification of specific target.
Step 1: 42C, 30 min
Step 2: 9 5C, 3 min Step 3: 95C, 30 sec
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
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).
Optimal specificity may be achieved by using the optimal annealing temperature for a given pair of primers in the PCR step of RT-PCR. The optimal annealing temperature for a pair of primers may be determined as follows. First, estimate the primer melting temperature (Tm) for each primer based on the number of G, C, A, and T residues in each primer, according to the following equation: Tm = [(G+C) x 4C] + [(A+T) x 2C]. Second, conduct RT-PCR with the pair of primers using an annealing temperature that is well below the lower Tm for the two primers (5 to 10C lower, for example). Third, increase the primer annealing temperature in increments of 2 to 3C in subsequent or parallel RT-PCR experiments, until optimal specificity is achieved. For best results, use pairs of primers that are well matched in terms of their Tm values.
Specificity may also be improved by use of relatively low amounts of primers in RT-PCR reactions. For relatively long (≥1.5 kb) RT-PCR products in particular, decreasing the concentration of each primer from 0.4μM to 0.075μM can have a positive effect on specificity (Fig. 4). Note that this approach tends not to work as well with relatively short (0.5 kb) RT-PCR products. In such cases low primer concentrations may result in decreased sensitivity and yield (Fig. 3).
Excellent yields may be obtained by optimizing factors such as product length (shorter products may result in higher yields), RNA concentration (more RNA may result in higher yields), and MgCl2 concentration (1.5mM to 2.5mM typically works well in one-step RT-PCR). One additional approach, which works well for some targets, is to increase the amount of RT-PCR enzyme mix in the reaction. For some relatively long (≥1.5 kb) RT-PCR targets, doubling t he amount of RT-PCR enzyme mix in the reaction can also dramatically increase the yield of product (Fig. 5).
Simple factors in RT-PCR, such as primer concentration, primer annealing temperature, and enzyme amount, have profound effects on sensitivity, specificity, and yield. Optimization of these factors is an excellent starting point for achieving desirable RT-PCR results.
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