The reverse transcription (RT) step of RT PCR for converting RNA to cDNA is critical for accuracy in quantification and for finding low copy messages. So you want to make sure that this step is performed with the highest efficiency but without having to optimize every single step.
To help you further in optimizing the RT process, here are some great enzymes and methods that can help you make a big difference in achieving high yields of difficult and low copy messages:
1. Choice of RT Primer
You have three choices of RT primer: oligo dT, random primers, or a gene specific primer. Many people use an OligodT primer so they can get full length copies of the mRNA. However, if the message is long (>4kb) or does not have a poly A tail (prokaryotic mRNA), then you will instead want to use random primers. Random primers will enable you to transcribe 5′ ends of long genes, but your cDNAs may not be full length copies of the entire gene. Typically 6-mers are used but using 8 or 9-mers can help increase the sizes of cDNAs since they will hybridize less frequently.
For qPCR of eukaryotic genes, the approach of using a mix of long random primers and oligo-dT has been shown to give the best result (see Figure 23, page 32). Two companies that provide this type of optimized primer mix with their RT kit are Qiagen QuantiTect Rev. Transcription Kit and the BioRad iScript Kit.
The third choice is a gene specific primer. Gene Specific primers enhance sensitivity by directing all of the RT activity to a specific message instead of transcribing everything in the mix. If you are performing a one-step RT-PCR, gene specific primers are used because the RT primer is also your reverse primer for the PCR step.
2. Secondary Structure in RNA
RNA secondary structure can be a problem in transcribing full length RNA. The RT enzyme can stop or fall off the template when it hits loop structures in the RNA.
It is difficult to know if your RNA will have secondary structure, but typically if the GC content of the gene is high, it can be an indicator that the RNA is going to be difficult to melt apart and may not be completely single stranded before the reaction.For this reason, a usual first step is a 5 minute 65C denaturation to relax the RNA.
However, another approach is to use an RT enzyme that allows for synthesis to occur at a higher temperature than standard RT. Thermoscript enzyme from Life Technologies is an example of a popular enzyme used for difficult templates. Alternatively, there are RT enzymes with higher efficiency at moving through secondary structure, even at standard RT incubation temperatures (37C-42C), and have been shown to give better results with GC rich templates. Examples of these are the Qiagen enzymes Omniscript and Sensiscript, and Takara Bio’s PrimeScript RT enzyme.
3. Removal of gDNA
Genomic DNA contamination in the RNA can be a cause of false positives in the final PCR. There are many ways to remove gDNA, such as DNase treatments during an RNA Prep or DNase treatments after a prep (such as the Turbo DNA-Free Kit by Ambion).
However, a better way to get around having to enzyme treat your precious RNA is to design primers that cross an intron or an intron-exon boundary. Using this method, no amplification can occur in DNA (or amplification of DNA results in a different sized band.) One problem to look out for is pseudogenes. A pseudogene is a DNA copy of the spliced mRNA inserted into the genome. Primers designed to RNA only will still amplify peudogenes.
For prokaryotic RNA which has no introns, genomic DNA removal becomes critical for accurate gene expression assays. Enzymatic removal is the only choice and can be performed during the prep as described above, and for extra assurance that it has been removed, the Quantitect Rev. Transcription Kit has a gDNA wipeout buffer that removes any residual DNA before starting the RT and does not require heat or EDTA inactivation. This enzyme is recommended for qPCR because cDNAs may not be full length.
PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio) is a reverse-transcription kit for real-time RT-PCR (RT-qPCR) that includes a genomic DNA elimination reaction. cDNA synthesis from RNA can be achieved without loss in a rapid reaction that is complete in less than 20 minutes.
4. Check the RNA Integrity
RNA quality will have a big impact on the results of cDNA synthesis. And batch to batch variation in RNA quality will lead to inconsistent results. Before performing an RT, you should check the quality of the rRNA bands. One way to do this is to run an agarose gel and check it visually. Intact eukaryotic RNA should show a 28s and 18s rRNA with the larger band looking close to double in intensity compared to the smaller. If the intensity is about the same, it is still ok.
What you don’t want to see if heavy smearing around the bands and especially low in the lane. This is degradation. If you don’t have enough sample to use on a check gel, a more accurate way of determining RNA quality is to use the Agilent BioAnalzyer. The BioAnalyzer provides you a visual representation of the RNA and calculates an RNA Integrity Number (RIN) to give you a quantitative measure of quality. A perfect score is a RIN of 10. RINs of 8 are ok. Below RINs of 7 and the RNA may have enough degradation to cause some problems detecting rare messages or providing consistent results.
They offer two different chips; one for detecting nanograms and one for picograms of RNA. This system is very expensive and so are the chips but if you have a core molecular biology lab or a microarray lab at your institution, they will likely have one of these instruments and can run samples, usually for a small fee.
For a fun and interactive presentation on RNA quality, check this out from Ambion.
5. Quantitating the RNA
Besides knowing the integrity of the RNA, accurate assessment of the yield is important as well. The accuracy of the yield can be affected by several things; accuracy of your measuring instrument, contamination with DNA, contamination with salts, and level of degradation. For measuring the yield, I prefer to use UV quantification using the Nanodrop.
This instrument does not require dilution of the sample and has a very wide range for measuring RNA. In our experience it can accurately read down to 10ng/ul. Conventional UV spectrophotometers with large cuvettes should be avoided for RNA because large volumes are needed for measuring samples. The downside of UV quantification is that genomic DNA will also be measured in the sample and if salts or phenol left over from the prep are contaminating the RNA, then there can also be added absorbance giving a false higher reading of the RNA.
Another way to measure RNA yield that gets around the problems of DNA and salt contamination is to use fluorescent dyes. Ribogreen is an RNA specific dye that can be used to measure yields using fluorescence. Nanodrop now has an instrument that can be used with Ribogreen.
6. Two-Step or One-Step RT-PCR
Whether to use a one-step or two-step RT-PCR is a question asked by all scientists. Shoba gave us some good advice on this topic for real-time PCR. There are advantages and disadvantages of each. With a one-step RT-PCR, a sample transfer step is eliminated, eliminating a potential source for contamination of controls. And with a one-step RT-PCR, because the primer is gene-specific, the sensitivity may be higher.
With two-step RT-PCR, a larger batch of RNA can be converted to cDNA and then aliquoted into many reactions. Two-step RT-PCR allows for greater choice in primers and also allows you to use the same batch of RT for analysis of multiple genes. This will help eliminate a potential contributor of variation when comparing different genes from the same source.
Regardless of which you choose, one-step or two-step RT-PCR, once you choose a method for your study, you need to stick with it. If you are performing qPCR, you will have a difficult time comparing data between the methods. If you just need a yes/no answer or are going to clone the cDNA you amplify, then switching back and forth won’t make a difference.
The reverse transcription process is influenced by many factors so to have the best results, it helps to design a strategy specific to each project. A method optimized for one project may not fit for another. It all depends on the type, level, and source of the RNA.
A thorough evaluation of strategies before you start will save you far more time in the long run.
Do you have any RT tips or questions you’d like to share…?