( ted. Reactions and primers are describe...)Simple Approaches for Optimization of RT-PCR

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
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