RT PCR

Overview of Assay

RNA oligonucleotides from cells and viruses can be amplified for detection using the methods of Reverse Transcription and Polymerase Chain Reaction (RT-PCR).  In a first step, a complementary strand of DNA (cDNA) is synthesized using a primer and reverse transcriptase.  The cDNA is then amplified using standard PCR techniques.  This can be accomplished by taking an aliquot from the reverse transcription reaction and adding a DNA-dependent DNA polymerase (e.g. Taq) and other reagents and then doing PCR.  That method is commonly called "Two-step RT-PCR", or "Two-tube RT-PCR".  Alternatively, all reagents, including both enzymes and gene-specific primers can be included in a single tube in order to do both reverse trancription and PCR without the need to open the tube between those reactions.  That method is commonly called "One-Step RT-PCR" or "One-Tube RT-PCR".  Both methods can include primers for a single target or for multiple targets (i.e. multiplex RT-PCR).

Common Problems

The majority of PCR is now done using a "hot-start" method to keep Taq inactive until it reaches reaction temperatures.  In contrast, the vast majority of reverse transcrption to date has been done without hot start even though reverse transcriptases have significant activity at room temperature and even below.  Due to this activity at low temperature and the high stability of DNA-RNA hybrids, primers intended to be gene-specific may hybridize with undesired targets that are partially complementary, leading to non-specific amplification.  This problem is increased substantially when multiple primers are included for multiplex RT-PCR.

How ThermaStop-RT Overcomes Common Problems

ThermaStop-RT is a simple-to-use reagent for One-Step or Two-Step RT-PCR initiated with gene-specific primers.  ThermaStop-RT interacts with reverse transcriptase at low temperatures to inhibit enzyme activity that can lead to non-specific products.  By eliminating these errors, ThermaStop-RT significantly increases the yield of the desired specific products and increases assay sensitivity.  Reverse transcriptase activity increases at temperatures above 45oC, enabling transcription of the desired RNA sequence.

Single-target-sequence (monoplex) RT-PCR

Approximately 1,000 copies of an RNA sequence from Hepatitis C Virus (HCV) Genotype 2b Armored RNA® [link: http://asuragen.com/wp-content/uploads/2016/04/Armored-RNA-Catalog-Rev-08.16_Final.pdf ] in either the presence or absence of ThermaStop-RT was reverse transcribed using SuperScript® III (Invitrogen) and amplified in a non-symmetric PCR using Platinum™ Tfi polymerase (Invitrogen).  Both one-step and two-step RT-PCR protocols were tested.  Figure 1 shows the end-point fluorescent signals from a hybridization probe.  The results indicate that for either one-step or two-step RT-PCR, over 3 times as much of the HCV amplification product was generated in samples containing ThermaStop-RT.

A large decrease in the amount of non-specific PCR product is also observed in samples containing ThermaStop-RT  Figure 2 shows the SYBR Green fluorescence derivative from the post-PCR melt of the two-step RT-PCR experiment above.  Samples containing ThermaStop-RT (purple lines) have a single melt peak at 85oC detecting the melting of the HCV-specific amplification product.  In contrast, samples without ThermaStop-RT (blue lines) have a much smaller melting peak at 85oC, consistent with reduced specific amplification, and have additional, non-specific product peaks.

Figure 1.  ThermaStop-RT (TS-RT) greatly increases probe signals for detecting Hepatitis C Virus (HCV) RNA sequence using either two-step or one-step RT-PCR.

Figure 2.  Melting analysis following two-step RT-PCR of an HCV RNA sequence detects a single, specific product in samples with ThermaStop-RT (purple lines). Non-specific products are detected in samples without ThermaStop-RT (blue lines).

Co-amplification of two RNA targets, including one with high secondary structure

A test was developed for the detection of a viroid that infects palm trees.  The viroid is an infectious, circular RNA that has extremely high secondary structure, posing a challenge for RT-PCR.  A synthetic RNA with the viroid sequence was generated using in vitro translation.  The mitochondrial nad5 transcript, which is trans-spliced from different regions of the mtDNA was used as a control for successful RT-PCR in the assay.  The viroid RNA was serially diluted and mixed with a constant concentration of palm leaf RNA.  One step RT-PCR in the presence or absence of ThermaStop-RT was done using non-symmetric gene-specific primers, PrimeScript reverse transcriptase (Takara) and Platinum Taq polymerase (Invitrogen).

Figure 3 shows the relative fluorescence signals from the viriod probe following RT-PCR.  The signals, which reflect the quantity of amplification product of the reaction, are significantly higher in samples with ThermaStop-RT.  That difference is highest in samples with low RNA concentrations.  Gel electrophoresis of the RT-PCR products of samples with only palm leaf RNA, only viroid RNA, or both is shown in figure 4.  Samples without ThermaStop-RT show faint viroid product bands and high levels of non-specific products while only the strongly stained specific product bands are detected in samples with ThermaStop-RT.

Figure 3.  ThermaStop-RT increases probe signals following one-step RT-PCR in samples with serial dilutions of a viroid RNA sequence plus a constant concentration of palm leaf RNA.  The palm mitochondrial nad5 transcript was co-amplified in all samples.  The improvement with ThermaStop-RT is highest in samples with low RNA concentrations.

Figure 4.  ThermaStop-RT dramatically reduces non-specific amplification in a one-step RT-PCR for a pair of RNA targets.  Five samples without and five samples with ThermaStop-RT show (left to right) amplification of the mitochondrial nad5 mRNA from coconut palm leaves, synthetic coconut palm viroid RNA, or co-amplification of both nad5 mRNA and each of the three lowest serial dilutions of the viroid RNA.  The specific 152- and 127-base pair bands are the only products detected in samples with ThermaStop-RT.  Samples without the reagent have lower levels of those products and high levels of non-specific product.  Bands on the left are from a quantitative DNA ladder (100-500 base pairs).