Real-time PCR, or quantitative PCR (qPCR), has quickly become instrumental for quantifying DNA and for quantifying RNA in gene expression studies by reverse-transcription qPCR (RT-qPCR). But the quality of the results strongly depends on one of the preparatory steps: synthesis of cDNA. For example, in gene expression analysis, the starting RNA sample must be converted into cDNA before qPCR can begin. Yet many researchers do not realize the implications of this step in the process.
Important variables to consider in setting up the cDNA synthesis step include sample contamination with genomic DNA, inhibitors from the prepared sample, enzyme fidelity, primers and sequence bias or content of the starting material. “When we talk to RT-qPCR researchers, we find that they pay less attention to this most critical step—cDNA synthesis,” says Ramesh Sathiyaa, product manager for PCR reagents at Bio-Rad. “Instead, they may be focused on PCR aspects, such as Cq values and fluorescence. While this may all be very valid, if you put garbage in, you’ll get garbage out. One of the most important points is RNA sample quality.” Highlighted here are examples of several factors, such as synthesis capacity and enzyme fidelity and efficiency, that researchers are concerned with today.
Larger capacities, longer targets, and broader temperature ranges
In some respects, bigger is better in cDNA synthesis today. People are looking for the ability to use larger starting samples in a synthesis reaction, to amplify longer targets and to work over a broader temperature range in the reaction. Bio-Rad’s newest cDNA synthesis kit addresses the capacity demand. The company claims that its iScript Advanced cDNA Synthesis Kit for RT-qPCR has the highest capacity of starting RNA (7.5 µg) on the market today, with a wide linear dynamic range (100 fg – 7.5 µg) in one 20 µl reverse-transcriptase reaction. “Researchers using the iScript Advanced cDNA Synthesis Kit for RT-qPCR can get more qPCR data points—up to nearly 1,000 target genes—from one 20 µl reverse-transcription reaction,” says Sathiyaa. “Generating all the data points from a single reverse-transcription reaction also significantly reduces inter-assay variability and saves money.”
Bio-Rad recommends optimizing the limits of starting RNA amounts. “Some researchers use the upper limit for input RNA amount per product recommendations without prior optimization,” says Sathiyaa. But optimizing can prevent wasting sample RNA. “If they’re working with medium or high abundant target genes, and they use a sensitive and efficient reverse-transcription kit, they may be able to use considerably less RNA and still get accurate quantitation. This not only saves RNA sample, in some cases, it won’t saturate the signal allowing detection of small fold changes.” Additionally, Life Technologies’ SuperScript® VILO™ cDNA Synthesis Kit is designed to give larger yields and linearity over a wide range of starting sample material: up to 100 µl per reaction.
Longer targets and broader temperatures are addressed by Agilent’s AffinityScript QPCR cDNA Synthesis Kits, which contain AffinityScript Reverse Transcriptase, an enzyme suited to both of these jobs. Additionally, the kit is sensitive enough to aid in cDNA synthesis of traditionally difficult samples, such as low abundance RNA, small amounts of starting samples or high-GC-content samples. Traditionally, RT enzymes had trouble producing good yields with high sensitivity across a wide temperature range (instead, researchers split the reaction into separate parts conducted at different temperatures). Agilent’s AffinityScript Multiple Temperature cDNA Synthesis Kits can help avoid this extra labor. “Many customers currently must change, or optimize, their reverse transcriptase for different samples and applications based on the target needs and results achieved,” says Laura Mason, global product manager for genomics at Agilent Technologies. “Instead of utilizing multiple different RTs and RT conditions, customers would get the advantage of using a single RT across all their samples with superior sensitivity and yields even on longer targets (linear detection from 3 pg to 3 µg total RNA).”
Higher-fidelity reverse transcriptase
Unfortunately, the fidelity of RT enzymes leaves something to be desired. “Reverse transcriptases exhibit significantly higher error rates than known DNA polymerases, introducing errors at frequencies of one per 1,500 [to] 30,000 nucleotides during cDNA synthesis, and are the major contributors of errors in RT-PCR, particularly with ultra-high fidelity PCR enzymes,” says Mason. “These RT-introduced errors are then amplified exponentially in further steps, and as a result customers must spend a significant amount of time and effort identifying and correcting errors after RT-PCR.”
One solution is temperature—fidelity of RT enzymes is generally improved at higher reaction temperatures (50 to 60°C). “The recent development of thermostable reverse transcriptase enzymes has opened entirely new possibilities to optimize reverse-transcription reaction conditions in this temperature range,” says Jakob Stenman, researcher at the Institute for Molecular Medicine, Nordic EMBL Partnership for Molecular Medicine, and the University of Helsinki. “RT enzymes with reduced activity at temperatures below 40°C, as well as development of heat-activated enzymes, will enable utilization of hot-start reverse-transcription protocols that can be very beneficial in many situations.” Life Technologies’s Platinum® Quantitative RT-PCR ThermoScript™ One-Step System uses a cloned avian reverse transcriptase with high thermal stability (up to 70°C), which can be used to measure mRNA from small numbers of cells and can detect as few as 10 molecules of RNA template, according to Life Technologies. Additionally, Roche Applied Science offers the Transcriptor line of cDNA synthesis kits for use with its LightCycler system.
Agilent also has engineered the AccuScript RT to deliver better transcription accuracy in its AccuScript High-Fidelity cDNA Synthesis Kits. “AccuScript, a proofreading RT, synthesizes cDNA which contains three- [to] six-fold fewer errors and produces higher-quality, full-length cDNA up to 20 kb,” says Mason. “While originally less important for qPCR applications, higher-fidelity RTs are becoming more and more valuable with newer applications and emphasis on use with sequencing and expression analyses.”
Primer-independent reverse-transcription variability
Theoretically, designing primers for PCR enables the user to specify the end products that will be amplified. However, primer-independent cDNA synthesis also can contribute to the final amplification product, hindering quantitative analysis. “There are large target-specific variations in reverse-transcription efficiency, regardless of priming strategy,” says Stenman. “Some of this variation is obviously due to differences in priming efficiency, but a considerable portion of the obtained cDNA in most reverse-transcription reactions is a result of non-specific primer-independent cDNA synthesis, even when reverse transcription is performed with target-specific primers.”
Stenman says that primer-independent cDNA synthesis also confounds the ability to detect specific strands, because it results in generation of the undesired cDNA strand during reverse transcription. This phenomenon is thought to occur when the undesired strand becomes primed not by the intended primers but instead by secondary hairpin structures or other small nucleic acids. This occurs naturally already; for example, antisense transcription is important in gene silencing and in coding RNA expression.
Stenman and colleagues developed an RT-qPCR assay to assess how much variation is a result of primer-independent reverse transcription . The assay incorporates primers that modify the sequence of the primed cDNA transcripts by switching the nucleotides adenine or thymine with cytosine or guanine. “Performing reverse transcription in multiplex with target-specific primers gives an opportunity to control and also modify the reverse-transcription efficiency of different targets in the same reaction,” says Stenman. Following PCR amplification, the two types of PCR products (primer-dependent and primer-independent) can be detected with melting curve analysis by a 3° to 5°C change in melting temperature. “The efficiency of reverse transcription and the proportion of non-specific cDNA synthesis can be monitored separately for each of the targets, if the specifically primed cDNA transcripts are tagged with a sequence modification that can be detected during or after PCR amplification,” Stenman says. “This technique provides a simple tool for optimizing reverse-transcription reaction conditions.”
An exciting application of RT-qPCR is to quantify the gene expression levels within a single cell. For example, Epicentre’s MessageBOOSTER™ cDNA Synthesis Kit for qPCR gives researchers the sensitivity to amplify the total RNA from a few cells or even from one cell (about 10 pg RNA). This is possible because the kit amplifies the mRNA first and then converts the amplified RNA to cDNA. Epicentre says it is possible to detect even low-abundance transcripts from the total RNA sample of a single cell. Sathiyaa believes that current cDNA synthesis kits are not particularly efficient without the pre-amplification step but sees change on the horizon. “There are concerns that the pre-amplification step may introduce a bias, if the kit is not well optimized,” says Sathiyaa. “Advances in the future may allow researchers to remove the pre-amplification step for single-copy detection.”
1. Feng L, et al., “Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription,” Biotechniques, 52(4): 263-270, 2012.
The image at the top of this page is from Bio-Rad's iScript Advanced cDNA Synthesis Kit for RT-qPCR (see Extended Dynamic Range).