by Catherine Shaffer
Quantitative real-time RT-PCR (qRT-PCR) is a powerful tool for measuring gene expression which is rapidly replacing other methods such as the immunoassay, due to its ease, accuracy, and ability to multiplex. Real-time PCR permits the quantification of DNA transcripts during the exponential stage of amplification through the use of dyes such as SYBR® Green. The reaction conditions needed to provide a robust exponential amplification vary somewhat from the requirements for successful endpoint PCR. The ideal reaction will provide reproducibly linear results for amplification products over a broad range of starting concentrations of RNA.
An often overlooked aspect of expression profiling is the efficiency of conversion of RNA into cDNA prior to qPCR. Despite its widespread use, the efficacy and accuracy of cDNA synthesis is not well understood for a wide range of gene sequences with different levels of expression. Most cDNA synthesis kits designed for endpoint applications or for cDNA libraries may not produce accurate and quantitative results. The conditions of reverse transcription that produce full-length cDNA are not necessarily quantitative and have not been optimized for use with small quantities of RNA sample and quantitative measurements. Thus, it is difficult to get good quantitative results using these older technologies.
A number of vendors are now offering cDNA kits optimized for the specific needs of qRT-PCR. Quanta Biosciences offered one of the first cDNA synthesis kits optimized for qRT-PCR. Says Ayoub Rashtchian, PhD, CSO of Quanta Biosciences, "We basically started at the ground floor and studied all aspects of cDNA synthesis to develop cDNA synthesis reagents that convert RNA into cDNA with high efficiency and without sequence bias. The other goal was to make cDNA synthesis as easy as possible." Most available kits, especially those for making cDNA libraries, contained up to ten different components that needed to be kept separate.
Quanta's qScript™ cDNA SuperMix is the first commercially available system in which all components necessary for cDNA synthesis have been stabilized together and only require addition of sample RNA. qScript cDNA SuperMix is an easy-to-use format for 2-step RT-PCR. This one-tube SuperMix contains all components for cDNA synthesis including buffer, dNTPs, MgCl2, an optimized blend of random and oligo(dT) primers, RNase inhibitor protein, qScript Reverse Transcriptase and stabilizers – all you need to add is RNA. Complete cDNA synthesis is achieved in only 40 minutes. Reducing the number of steps in the protocol not only decreases the workload for the technician, it reduces experimental errors by eliminating pipetting steps and improves the reproducibility of cDNA synthesis.
The value of optimizing the cDNA synthesis step is easy to show in the quality of the final results. Quanta's qScript kits yield consistently linear cDNA synthesis from 1 pg of RNA up to 1 ug. This is important because without linearity, the accuracy of quantification data can be compromised. It will be impossible to determine whether a variation is due to gene expression or an error introduced during cDNA synthesis. Says Rashtchian, "It's very important that regardless of the amount of input of the RNA, one gets representative results."
Other important considerations in qRT-PCR are the speed and efficiency of the reaction. Equipment is often shared among many users, so reducing the total reaction time allows more users to access the instruments. The AffinityScript qPCR cDNA synthesis kit by Stratagene, an Agilent Technologies Company reduces a typical reaction time from 30, 60, or 90 minutes down to just 15, for the cDNA synthesis portion of the reaction, and works over a wide temperature range (from 37 to 55°C). "You don't have to rely on doing reverse transcription at one individual temperature like 37 or 42 degrees," says Rachel Formosa, Product Line Manager for Genomics Reagents, Agilent Technologies. "Sometimes different RNA samples and gene targets you're looking at may be more difficult to work with than others. Some have secondary structure and the priming site may not be accessible. If you use a reverse transcriptase that works at a wide temperature range, you can increase the reaction temperature for more difficult targets. Higher temperature can also reduce the chances of mispriming."
The AffinityScript qPCR cDNA synthesis kit also zeroes in on the efficiency of RNA to cDNA conversion. The cDNA conversion efficiency of reverse transcriptases has been reported to be proportional to the RNA target levels. In other words, the lower the target abundance, the lower the conversion efficiency. The maximum conversion efficiency provided by AffinityScript qPCR cDNA synthesis kit significantly improves the sensitivity of the assay and its dynamic range, especially when transcribing very low abundance RNA targets.
Inihibitors in the reaction mix can sabotage cDNA synthesis, so an important part of optimizing a reaction mixture is dealing with inhibition. Applied Biosystems and Applied Biosystems business Ambion have given careful attention to inhibition of cDNA synthesis in their qRT-PCR products. The standard method for dealing with inhibition is through the process of dilution. However, Ambion's Fast Cells-to-CT™ kit includes proprietary components that prevent inhibition. Ambion researchers developed the formula over several years. "We wanted to have perfect performance in terms of efficiency and tolerance of what we could put in there. We took the RT components and sample components and ended up tweaking them through a series of experiments, using ultimately proprietary methodologies and compounds...to make all of these components tolerant and compatible," says Christina Buchanan, PhD, Product Manager for Ambion.
Although it seems ideal to eliminate all of the "bad influences" from a cDNA synthesis reaction, some surprising results from Applied Biosystems suggest that the issue is a bit more complicated than that. For amplification of longer fragments, Applied Biosystems has found that leaving a bit of RNase H activity in the reaction mix might be beneficial. Says Paul Kotturi, PhD, product manager for Applied Biosystems, "Most Taqman® or SYBR assays are 100 base pairs or less. As opposed to the endpoint market, quite often you're trying to amplify complete transcripts... If you compare the enzymes that are in our one-step and two-step kits and cDNA synthesis kits to an enzyme that has completely eliminated RNase H capacity, our kits outperfrom the competition...RNase H activity may be desireable if you're creating longer fragments."
It's clear that paying attention to the cDNA synthesis portion of a qRT-PCR experiment is well worth the time and effort in terms of the accuracy and sensitivity of the final results, and that the requirements for a qRT-PCR reaction vary drastically from endpoint PCR. Additionally, kits that decrease the number of preparatory steps can enhance the accuracy of results as well as saving time at the bench.
More Representative, Sensitive, two-step qRT-PCR
Use the Transcriptor First Strand cDNA Synthesis Kit from Roche to reverse transcribe RNA (mRNA, total RNA, viral RNA, and in vitro-transcribed RNA) for a variety of applications, including studying gene expression levels via two-step RT-PCR, generating cDNA libraries, and cloning genes of interest. Transcribe a variety of templates, even the most difficult (e.g., GC-rich RNA with high secondary structures), and obtain accurate linear quantification over at least a 108 -fold range of input RNA ( in vitro transcripts).
