One step vs. two step RT-PCR. Two step RT-PCR protocols typically produce higher yields of cDNA than one step procedures. This is because each step can be optimized separately, whereas conditions compromised to be compatible with both reverse transcription (RT) and PCR must be used for one step protocols. The two step reaction is also more versatile, in that it allows a single RT reaction to be used for multiple PCRs. Finally, reagents are conserved in the two step reaction because it is done in a smaller volume. One step RT-PCR generally works well for amplifying targets that are reasonably abundant, however we recommend using the two step protocol for most applications.

Random primers vs. oligo(dT) primers. Random oligonucleotides or oligo(dT) primers are typically used to prime reverse transcription reactions. The greatest yield of RT product is usually obtained by using short random oligonucleotides to prime the RT reaction. For this reason random primers are recommended when the template is limiting because final RT-PCR yield may be higher. For cDNA library construction or cDNA labeling applications, oligo(dT) primers are almost always used to prime cDNA synthesis to ensure that poly(A) containing mRNAs are reverse transcribed. Both types of primers are included in Ambion's RETROscript^™ First Strand cDNA Synthesis Kit.

Endogenous priming can be a concern. A significant amount of non-specific priming occurs during RT reactions performed under standard conditions. This is due to the presence of fragmented RNAs and small RNA molecules in the RNA sample that can also serve as primers for RT (in addition to those added exogenously). If your RT application requires initiation from a specific sequence, as with 3' RACE, anchored- or T7 promoter oligo(dT) priming, or labeled cDNA synthesis using gene specific primers, endogenous priming can lead to aberrant results.

Use high quality RNA. The overall quality of a total RNA preparation can be assessed by electrophoresis on a denaturing agarose gel. Ribosomal RNA (rRNA) bands should be fairly sharp and intense with the upper 28S (or 23S) rRNA band being about twice as intense as the lower 18S (or 16S) rRNA band. A diffuse smear of mRNA can sometimes be seen migrating between the ribosomal bands. Alternatively, samples as small as 10 ng can be run on an Agilent bioanalyzer and RNA quality evaluated by quantitation of the two rRNA bands.

Remove genomic DNA from RNA for RT-PCR.
RNA that will be used in RT-PCR should be completely free of genomic DNA contamination. The presence of even trace amounts of DNA can generate false positive signals during PCR. No RNA isolation method will consistently produce DNA completely free of RNA unless a DNase treatment step has been incorporated into the procedure. For more information, read "Avoiding DNA Contamination in RT-PCR" or "Methods to Remove DNA Contamination in RT-PCR". Ambion also provides the DNA-free^™ DNase Treatment and Removal Reagents for treating RNA that will be used in a subsequent amplification step.

Total vs. poly(A) RNA. Use up to 5 µg of total RNA per RT reaction. If the RT yield is still low, it may help to use poly(A) RNA rather than total RNA. The optimal amount of poly(A) RNA should be determined empirically. Keep in mind that the copy number of an average transcript in 5 µg of total RNA is roughly similar to that present in 160 ng of poly(A) selected RNA.

Remove RT inhibitors. Contaminants can usually be removed by phenol extraction and re-precipitation of the RNA, followed by a 70% ethanol wash. To remove all traces of alcohol, respin the tube for a few seconds and remove the residual alcohol with a drawn out glass pipette, or a fine bore pipet tip.

Test for RT inhibitors. If you have a low yield of RT product, check for RT inhibitors in the RNA. Possible inhibitors of RT reactions include RNase and contaminants such as guanidinium, proteinase K, or alcohol carried over from the RNA isolation procedure. You can test for RT inhibitors by doing a mixing experiment. Set up three RT reactions, one containing a control RNA sample that has reverse transcribed well in the past, one containing the RNA sample with which you are currently having problems, and one tube with these two templates mixed. Add a trace amount of radiolabeled nucleotide to the reactions, incubate them, and analyze the results on a denaturing polyacralymide gel (8M Urea, 5% acrylamide). In effective RT reactions, you should see a smear that runs larger than ~800 bases corresponding to the majority of the first strand cDNA. This should at least be evident for the tube containing the control RNA (alone). When the two mixed templates produce less/smaller cDNA than the control alone, there is most likely a contaminant in the RNA and the sample should be further purified. Ambion's RT-Check^™ RT-PCR Sample and Procedure Control Kit provides reagents and control RNA sample and primers for testing problematic RT-PCR.

Avoid RNases! Trace amounts of ribonucleases can sabotage RT reactions. Since RNases are present throughout the laboratory environment as well as within the tissue or cell sample from which RNA is purified, it is important to take steps to prevent and eliminate RNase
contamination. Ambion offers a complete line of products designed to detect and eliminate RNases. See www.ambion.com/basics/
rnasecontrol for more information.

Analyze RT results. To analyze RT results a trace amount of radiolabeled nucleotide needs to be incorporated into the cDNA. A 10th of the reaction is then size fractionated on a denaturing polyacrylamide gel (8 M Urea, 5% acrylamide). In a robust RT reaction using either oligo d(T) or random primers, you would expect to see a smear of differing sized products. However if you are using a gene specific primer, you will see the majority of the product running as a discrete band.

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