by Jeffrey M. Perkel
Genomic DNA often is likened to a genetic "blueprint." But it might more accurately be compared to an encyclopedic cookbook. This cookbook contains "recipes" for whipping up every protein or regulatory RNA in the body, but not every cell needs to make every recipe. Neurons express a different subset of genes than do skin cells, for instance. The collection of recipes a cell makes is a molecular signature for that cell as it exists at any given moment, whether defined by developmental status, cellular identity, stress, drug treatment or disease.
Naturally, researchers are keen to study the RNA complements of different cells to see how they change over time or differ from tissue to tissue, for instance. But there's a catch: RNA is a molecular pain-in-the-pipetting hand. "RNA is inherently unstable," says David Kerry, a product manager at Life Technologies.
Enter complementary DNA, better known as cDNA: a DNA copy of an RNA molecule. Used chiefly for expression analysis (via real-time PCR, microarrays or sequencing), cloning and library construction, cDNA synthesis creates a more stable, more malleable template upon which to work.
cDNA synthesis exploits the viral enzyme reverse transcriptase (RT), which uses mRNA as a template to drive DNA synthesis, a step called reverse transcription. Researchers can choose from among a wide variety of commercial cDNA synthesis kits. Some are designed strictly for first-strand cDNA synthesis; others make double-stranded molecules. Some couple cDNA synthesis and quantitative real-time PCR in a single reaction; others separate the steps. Below, we review some of the key criteria users should consider to choose the cDNA synthesis kit that best meets their needs.
cDNA synthesis requires an oligonucleotide primer to drive reverse transcription. Three options are available: oligo-dT, random primers and gene-specific primers. Oligo-dT primers are complementary to the polyadenylated tail of most eukaryotic mRNAs. Thus, they enable users to specifically copy all mRNA transcripts and make full-length copies at that. "If you're cloning the cDNA, then you would likely start with oligo-dT as a primer strategy because you want a full-length cDNA," says Viresh Patel, marketing manager in the gene expression division at Bio-Rad Laboratories.
On the other hand, says Miltenyi Biotec senior scientist Stefan Wild, "If you are doing RT-PCR, it's not so important to get full-length [products]." Indeed, as RT-PCR amplicons usually range from 70 to 200 bases or so, they are compatible with all three priming strategies, including random priming.
Random primers can prime from anywhere and thus typically produce shorter fragments than does oligo-dT, but random primers also offer better coverage across the length of long transcripts (as they eliminate the potential bias caused by incomplete oligo-dT-driven synthesis produced by poor polymerase processivity or secondary structure). In addition, random primers can prime off non-messenger RNAs (such as non-coding RNAs). Some kits combine oligo-dT and random primers. For instance, the Miltenyi Biotech µMACS™ SuperAmp™ kit uses both primers to ensure relatively even coverage of a transcript pool prior to cDNA amplification and labeling for microarray analysis.
Gene-specific primers, as their name suggests, enable users to fish out and copy only the transcript of interest from an RNA pool. The approach, says Kerry, provides a simple way to measure the expression of a handful of genes; but that only works if researchers are certain they never will need to reanalyze the sample again later. "If you use gene-specific primers to make cDNA, you cannot go back and look at something else," he says.
One-step vs. two-step RT-PCR
Another key differentiator in cDNA synthesis kits is how they interface with downstream PCR—the "top application" for cDNA synthesis, according to Patel. So-called "one-step" kits include both steps in a single reaction—RNA in, RT-PCR results out. Two-step kits separate the steps.
The advantages of one-step kits, says Brigitte Hloch, international marketing manager at Roche Applied Science, which offers both kit types, include decreased time and increased sensitivity and specificity. One-step systems (such as the company's Transcriptor One-Step RT-PCR Kit) are also convenient, at least if users are only interested in a relatively small number of transcripts and can reduce the risk of contamination. "If it is important for you to reduce the risk of contamination, for example in a virology department, then I would always recommend to go with one-step RT-PCR," says Hloch.
Two-step systems, though, like Roche's Transcriptor First Strand cDNA Synthesis Kit, enable reaction optimization and "give maximal flexibility for RT-PCR on real-time instruments and conventional thermal cyclers," Hloch says, in that researchers can perform multiple RT-PCR reactions from a single RNA sample. That, says Wild, means researchers can also achieve less reaction-to-reaction variability with two-step reactions. Plus, Wild adds, the cDNA sample can be stored for subsequent reanalysis. (Miltenyi Biotec's µMACS One-Step cDNA Kit is a two-step system.)
Reverse transcriptase is the key enzyme in cDNA synthesis. But all RTs are not the same. Some RT enzymes, for instance, derive from avian myeloblastosis virus (AMV) and others from Maloney murine leukemia virus (MMLV). According to Promega product manager Amy Hendricksen, the former is better at handling difficult secondary structures in RNA templates, and the latter exhibits better processivity. Promega offers both forms of RT; the company's one-step AccessQuick RT-PCR system is based on AMV, whereas its GoScript polymerase derives from MMLV.
Another variable is RNAse H activity. According to Patel, RNAse H-positive enzymes (such as Bio-Rad's iScript) destroy the RNA as it's copied. "For RNA expression analysis, that is a smarter choice because you don't bias the population," he says. "You need to preserve the quantity of the original material." On the other hand, for cloning and "other non-quantitative applications," an RNAse H-deficient enzyme is preferable, as it can enable multiple priming rounds off each transcript and increase cDNA yield.
RNAse H status also matters if you are working with particularly lengthy mRNAs. "You don't want [RNAse H] activity if you are making long transcripts," says Kerry. "If you are trying to make cDNAs longer than 5 kb, it will degrade the RNA during synthesis." Life Technologies' SuperScript III enzyme is RNAseH-deficient, he says.
Finally, there is RT's notorious error rate. According to Hloch, the RT step in RT-PCR produces about 10 times more errors than the PCR step (which itself is error-prone). The product literature for Roche's Transcriptor High Fidelity cDNA Synthesis Kit highlights this problem; according to this document, the error rate for a normal MMLV reverse transcriptase is 1.37 x 10-4 errors per base, compared with 1.47 x 10-5 for Taq DNA polymerase.
Although this may not matter for most RT-PCR and microarray-based readouts, it can be especially problematic during cloning, sequencing and probe-based RT-PCR. Roche's Transcriptor High Fidelity Reverse Transcriptase formulation, with an error rate of 1.98 x 10-5, combines "a new recombinant reverse transcriptase and an enzyme conferring proofreading activity," according to the product literature, yielding "an 8.7-fold higher fidelity compared to conventional RT-PCR."
Researchers also should consider the following when selecting a cDNA synthesis system:
1. Application. The key decision point, says Patel, is your intended application. Do you plan to clone the cDNA, or are you doing expression analysis?
2. Kit format. Not all systems are equally user-friendly. Some, says Patel, can include "10 or 12 separate components." Others come as pre-mixed formulations—just add primer and template. Bio-Rad's iScript Reverse Transcription Supermix for RT-PCR includes everything in one tube, says Patel. "Just add RNA plus water, and the reaction is ready to go," he explains.
3. Single-strand or double-stranded output. For gene-expression applications, first-strand cDNA synthesis is all that's needed, says Patel. But for some applications, especially cloning and library construction, you'll need a double-stranded product, so look for a system that can generate that. (Such systems will include, in addition to the RT, a DNA-directed DNA polymerase to drive the second-strand synthesis.)
4. Scalability. If throughput is an issue, you might want to consider a format that is relatively scalable. Miltenyi Biotec's approach, relying on micro-scale beads and a strong magnetic field, can be automated easily, says Wild. That's because the process occurs entirely on a single column in a magnetic field, with no tube changes required and no sample movement. The company's two-step cDNA synthesis kits are available in both single-reaction and 96-well formats.
5. Sensitivity. Not all kits are equally adept at handling sparse samples, so check the system's specifications. Wild says Miltenyi Biotech "specializes in rare cell isolation," processing the RNA from as few as five cells in its standard cDNA synthesis platform and from "as little as one cell" on its SuperAmp cDNA amplification system.
6. Thermostability. "RNA often has a lot of secondary structure," says Kerry. "The higher the temperature you can do the RT at, the more likely the stem-loops will relax and your enzyme can push through." Life Technologies' SuperScript III works at up to 55oC, he says.
With so many options from which to choose, it's easy to get overwhelmed. Fortunately, in many cases, you really can't go wrong. Just remember to keep things in perspective: cDNA synthesis is almost never the goal of an experiment, but rather the beginning. "Making [RNA] into cDNA enables you to analyze it," says Kerry. "It's a means to an end."
The image at the top of this articled is Bio-Rad's iScript™ Reverse Transcription Supermix for RT-qPCR