Single-Tube Analysis Can Help You Avoid These 5 Common RT-PCR Challenges

The newest color-changing buffers and universal annealing can help you avoid some of the most common RT-PCR problems

RT-PCR (reverse transcription-polymerase chain reaction) is a popular method for revealing target gene expression. This relatively simple and cost-effective process is sensitive enough to detect RNA from even very small samples.

Now, our newest one-step RT-PCR system with universal annealing and colored buffers can make things even simpler and faster.

Read on to learn how Invitrogen^TM SuperScript^TM IV UniPrime^TM One-Step RT-PCR can help you avoid some of the most common RT-PCR challenges.

1. Not enough time

In the fast-paced, time-strapped lab, one-step RT-PCR can be a researcher’s best friend.

With a one-step approach, reverse transcriptase and DNA polymerase are pre-mixed into a single tube, enabling you to simplify setup and minimize pipetting steps to bypass cDNA isolation. This can enable a faster workflow and help decrease the chance of contamination.

But one-step RT-PCR itself is not new. Where the newest SuperScript IV UniPrime One-Step RT-PCR System really shines is in doubling down on efficiency and reaction speed by incorporating enzymes with particularly high processivity. It can knock out the RT step in as few as 10 minutes, followed by a 30 s/kb extension time – one of the shortest one-step kit protocols on the shelves today.

The system also neatly accommodates complementary time-saving approaches like multiplexing and automation to enable maximum productivity.

Summary of one-step versus two-step RT-PCR

2. Pipetting errors

With repetitive tasks like pipetting, it’s easy to lose track of which tubes you’ve already mixed.

The SuperScript IV UniPrime One-Step RT-PCR System arrives ready-to-go with premixed components, so reaction setup is already simplified. But the system also introduces an innovative color-change buffer approach to help you visually track your process in the run setup and ultimately help prevent simple pipetting errors that can derail hours of work. Red and blue tracking dyes in each of the reaction components form purple when successfully combined.

It’s fun to watch, but more importantly, it keeps your workflow running smoothly.

3. Wrong annealing temperature

One of the most common errors in RT-PCR is using the wrong annealing temperature.

Annealing temperatures are calculated from the melting temperatures (T_m) of PCR primers. The recommended melting temperature for a set of PCR primers is usually between 55-70° C and within 5° C of each other. But differences in paired primer sequences, lengths, and composition can make it difficult to hit that 5°C “Goldilocks” range. A suboptimal annealing temperature can lead to nontarget binding and drastically affect the yield and specificity of your reaction.

The SuperScript IV UniPrime system allows you to skip the T_mcalculator completely, with universal annealing at 60° C and no sacrifice in yield. As a nice bonus, no calculations can enable a faster workflow.

4. Inhibitor issues, small sample size, or low reaction performance

You don’t need to sacrifice performance to take advantage of one-step RT-PCR, even with challenging samples.

Inhibitory compounds are common in RNA samples, even after purification. The enzymes in SuperScript IV UniPrime One-Step RT-PCR system are engineered for high processivity and increased template affinity, which means that even in the face of routine inhibitors and sample impurities, they can perform well.

The system can also handle low-abundance targets beautifully for a one-step reaction, with high sensitivity and reliable detection down to just 0.01 pg RNA input. The increased sensitivity is owed to an innovative two-phase hot-start mechanism that prevents overlap of RT and DNA polymerase activity to help maximize reaction efficiency.

A gel revealing high sensitivity and reliable target detection from low amounts of input RNA in one-step RT-PCR

Figure 1. High sensitivity and reliable target detection from low amounts of input RNA. A 0.43 kb fragment was successfully amplified using serial dilution from 1,000 ng to 0.01 pg of Invitrogen Universal Human Reference RNA (UHRR) and the SuperScript IV UniPrime One-Step RT-PCR System. The molecular weight marker is the Thermo Scientific GeneRuler 100 bp Plus DNA Ladder, ready-to-use. NTC: no-template control.

5. Not scalable

Scalability is non-negotiable for many labs today.

The SuperScript IV UniPrime One-Step RT-PCR system was developed with high-throughput workflows in mind. The two-phase hot-start activation mechanism keeps the preassembled reactions stable at room temperature for extended periods – giving you ample time to complete dependent steps in a scaled-up workflow. For targets up to 3kb, you can achieve quality results even after reactions are left for 24 hours at room temperature. Longer-target reactions can be stored up to 4 hours.

The system is also compatible with both singleplex and multiplex applications.

Looking for more PCR Tips & Tricks? Check out our free, downloadable infographic.

Learn more about the SuperScript IV UniPrime One-Step RT-PCR system at thermofisher.com/ssiv-onestep»

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