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During gene expression, once the RNA transcript is reverse transcribed into cDNA, the cDNA becomes the actual template for the qPCR amplification process. As RNA is more fragile than DNA, it is not suitable for PCR amplification. Therefore, RNA must be converted into complementary DNA (cDNA) first. A reverse transcriptase enzyme catalyzes the process of cDNA synthesis by using RNA as a template.
Two methods are available for quantification of gene expression by RT-qPCR: “one-step” or “two-step” RT-qPCR. In both cases, RNA is reverse transcribed into cDNA, and the cDNA is then used as the template for quantitative PCR amplification. One-step or two-step refers to whether the reverse transcription and subsequent real-time PCR amplification are performed in the same or different tube.
In one-step RT-qPCR, cDNA synthesis and qPCR step occur in the same reaction. Two-step RT-qPCR uncouples cDNA synthesis and subsequent qPCR so they occur in separate reactions.
In one-step RT-qPCR, cDNA synthesis and qPCR are carried out inside the same reaction tube, in a common buffer, using gene-specific primers. Gene-specific primers direct cDNA synthesis and amplification of a specific target, and they typically anneal at higher temperatures compared to random primers. As a result, one-step protocols often use higher RT reaction temperatures than two-step workflows and can employ engineered or novel reverse transcriptase enzymes which tolerate higher reaction temperatures. Primary advantages of one-step reactions include minimal sample handling, reduced bench time, and closed-tube reactions, reducing the possibility of pipetting errors and cross-contamination. Therefore the one-step approach remains an ideal option for quantitating the same gene(s) repeatedly, which is particularly valuable in high-throughput applications and in diagnostic settings.
The quality and scarcity of RNA samples are important considerations for one-step RT-qPCR. Where RNA is of low quality, and inhibitors are inconsistently present between samples, reaction efficiency can be impacted to varying degrees, reducing comparability between samples. In addition, because the original cDNA synthesis product cannot be saved after one-step RT-qPCR, additional aliquots of the original RNA sample(s) are required to repeat reactions or to assess the expression of other genes.
In two-step RT-qPCR, cDNA synthesis can be carried out using random hexamers, oligo-dT primers, and/or gene-specific primers. Reactions using random hexamers and/or oligo-dT primers produce cDNA that is a mixture of various RNA species in the sample. The cDNA synthesis reaction can be scaled up to accommodate higher RNA input, and extraction and precipitation steps can be used to concentrate and/or further purify the cDNA. Although the two-step workflow requires more time, it allows better control over the amount of cDNA template that is used for qPCR quantitation. Because only a portion of the cDNA product is used in the subsequent qPCR step, remaining cDNA can be stored for future use, or quantitating the expression of multiple genes from a single RNA/cDNA sample.
Two-step RT-qPCR uncouples cDNA synthesis and subsequent qPCR, allowing more options in the selection of RT enzymes separately from the qPCR reagents for amplification. This flexibility can be useful for controlling sequence representation, efficiency, and optimization of difficult RT-qPCR reactions.
The key drawbacks of two-step RT-qPCR are the extra open-tube step, greater number of pipetting manipulations, and longer hands-on time. The result is a greater possibility of variability and risk of contamination, and thus, two-step RT-qPCR is less amenable to high-throughput applications.
One-step RT-qPCR can be achieved either by using Thermus thermophilus (Tth) polymerase, a DNA polymerase with inherent RT activity, or by a two-enzyme system combining a reverse transcriptase with a thermostable DNA polymerase. Since Tth DNA polymerase is derived from a thermophilic bacterium, higher temperatures (>60ºC) can be used for the RT step, which can minimize secondary structure in high-GC-content mRNAs.
Accurate analysis of gene expression requires good reproducibility of reverse transcription reactions. The robustness of a reverse transcription is determined by the sensitivity, dynamic range, and specificity of the reverse transcriptase used. Reverse transcriptases from Moloney murine leukemia virus (MMLV) and avian myeloblastoma virus (AMV) are the most commonly used enzymes. When long or full-length cDNA transcripts are needed, MMLV reverse transcriptase and its derivatives are better choices than AMV reverse transcriptase due to their lower RNase H activity. It is has been observed, however, that RT reactions performed with RNase H– reverse transcriptases sometimes require subsequent RNase H treatment to effectively amplify certain templates and those at low concentration in the PCR.
As noted above, the reverse transcription step in two-step RT-qPCR can be performed using oligo(dT) primers, random primers, a mix of these two, or gene-specific primers. The choice of primers may influence quantification of your target gene. Gene-specific primers give less background priming than oligo(dT) or random primers. If you choose oligo(dT) primers for reverse transcription, you may want to place PCR primers close to the 3' end of the transcript to avoid loss of sensitivity due to truncated messages; this is especially important for longer transcripts. Oligo(dT) priming should be avoided if you are working with transcripts or species that have short poly(A) tails or lack them altogether. For the 5'-nuclease assay, we found oligo(dT)–primed cDNA to perform very well; however, random-primed cDNA performed equally well or slightly better for most sequences, and much better for some sequences. For most PCR applications, we have found best performance using iScript and iScript Select cDNA synthesis kits, which employ an MMLV RT.
Ultimately, a good reverse transcription (RT) kit is useful to produce a complete and representative cDNA copy of the mRNA.
The hallmarks of a good RT kit include: 1) a mixture of random hexamers and oligo(dT)s to assure complete coverage of the transcriptome; 2) RNase H to digest the RNA while the cDNA is synthesized, which minimizes bias in cDNA production preventing the Cq values from being skewed to lower and variable values that would be unrepresentative of the true target amount in each sample; 3) an RNase inhibitor to prevent degradation of the RNA prior to RT; 4) a highly robust RT enzyme that permits RT of the widely ranging concentration of transcripts populating each sample. iScript Reverse Transcription Reagents from Bio‑Rad contain a blended mixture of each of these components to assure cDNA that closely reflects the transcriptome.
Reliance One-Step Multiplex Supermix from Bio-Rad offers a good example of a high-performance RT enzyme combined with an advanced reaction buffer which is designed for one-step RT-qPCR. The supermix format delivers reproducible performance for multiplexed RT-qPCR, and can be used with challenging RNA templates, difficult targets or in the presence of PCR inhibitors.
Overview of Reverse Transcription
Reverse transcription begins when the viral particle enters the cytoplasm of a target cell. The viral RNA genome enters the cytoplasm as part of a nucleoprotein complex that has not been well characterized. The process of reverse transcription generates, in the cytoplasm, a linear DNA duplex via an intricate series of steps. This DNA is colinear with its RNA template, but it contains terminal duplications known as the long terminal repeats (LTRs) that are not present in viral RNA ( ). Extant models for reverse transcription propose that two specialized template switches known as strand-transfer reactions or “jumps” are required to generate the LTRs.
Reverse transcription of the viral RNA genome generates a linear DNA duplex. The positions of the R, U5, and U3 regions, the polypurine tract (PPT), and the primer-binding site (PBS) are indicated. Reverse transcription creates duplications of the U5 (more...)
Retroviral DNA synthesis is absolutely dependent on the two distinct enzymatic activities of RT: a DNA polymerase that can use either RNA or DNA as a template, and a nuclease, termed ribonuclease H (RNase H), that is specific for the RNA strand of RNA:DNA duplexes. Although a role for other proteins cannot be ruled out, and it is likely that certain viral proteins (e.g., nucleocapsid, NC) increase the efficiency of reverse transcription, all of the enzymatic functions required to complete the series of steps involved in the generation of a retroviral DNA can be attributed to either the DNA polymerase or the RNase H of RT. The process of retroviral DNA synthesis is believed to follow the scheme outlined in :
Minus-strand DNA synthesis is initiated using the 3′end of a partially unwound transfer RNA which is annealed to the primer-binding site (PBS) in genomic RNA, as a primer. Minus-strand DNA synthesis proceeds until the 5′end of genomic RNA is reached, generating a DNA intermediate of discrete length termed minus-strand strong-stop DNA (–sssDNA). Since the binding site for the tRNA primer is near the 5′ end of viral RNA, –sssDNA is relatively short, on the order of 100–150 bases
Following RNase-H-mediated degradation of the RNA strand of the RNA:–sssDNA duplex, the first strand transfer causes –sssDNA to be annealed to the 3′end of a viral genomic RNA. This transfer is mediated by identical sequences known as the repeated (R) sequences, which are present at the 5′ and 3′ends of the RNA genome. The 3′end of –sssDNA was copied from the R sequences at the 5′end of the viral genome and therefore contains sequences complementary to R. After the RNA template has been removed, –sssDNA can anneal to the R sequences at the 3′end of the RNA genome. The annealing reaction appears to be facilitated by the NC.
Once the –sssDNA has been transferred to the 3′R segment on viral RNA, minus-strand DNA synthesis resumes, accompanied by RNase H digestion of the template strand. This degradation is not complete, however.
The RNA genome contains a short polypurine tract (PPT) that is relatively resistant to RNase H degradation. A defined RNA segment derived from the PPT primes plus-strand DNA synthesis. Plus-strand synthesis is halted after a portion of the primer tRNA is reverse-transcribed, yielding a DNA called plus-strand strong-stop DNA (+sssDNA). Although all strains of retroviruses generate a defined plus-strand primer from the PPT, some viruses generate additional plus-strand primers from the RNA genome.
RNase H removes the primer tRNA, exposing sequences in +sssDNA that are complementary to sequences at or near the 3′end of plus-strand DNA.
Annealing of the complementary PBS segments in +sssDNA and minus-strand DNA constitutes the second strand transfer.
Plus- and minus-strand syntheses are then completed, with the plus and minus strands of DNA each serving as a template for the other strand.
Process of reverse transcription of the retroviral genome. (Black line) RNA; (light color) minus-strand DNAs; (dark color) plus-strand DNA. See text for a description of this process.
A more detailed description of these steps is presented below.