Have you ever been setting up your qPCR experiment and gotten stuck trying to decide which Reverse transcription, or RT, method to use? You’re not alone!
We’ve received quite a few questions about RT methods so I thought it might be a good idea to learn about what methods are out there and why choosing the right method matters.
Let’s take a look at our lab book
There are two primary approaches to reverse transcription for qPCR known informally as two-step or one-step methods. In the Two-step approach the first step is reverse transcription of the RNA to generate the 1st strand cDNA, or complementary DNA. At this point the cDNA can be stored or moved to the second step. In the second step the 2nd strand cDNA is generated and real-time detection is performed. The other method, known as one-step, is where the reverse transcription and amplification are performed together in the same tube. That’s how you get 1-step and 2-step.
Pretty self-explanatory nomenclature, right?
So how do you know which method to choose?
The two-step method is required when analyzing multiple targets in separate reactions using the same cDNA. For instance, one cDNA synthesis could be used to detect ten different gene expression targets. You can also store the cDNA for later use with the two-step approach, and this is the preferred method for use with limited sample quantities.
Conversely, when using the one-step approach, the cDNA cannot be stored and the experiment is limited to multiplex constraints when looking at multiple targets because everything occurs in that one reaction. However, this approach is faster and doesn’t require as much hands-on time. The one-step method is great for use with liquid-handling robotics which can reduce the chance for cross-contamination and pipetting errors. Useful for high-throughput gene expression analysis, the one-step method is also great for RNA virus detection, where specific detection can generally be accomplished in a single multiplex reaction and time to results is particularly important.
So now that you’ve decided which approach to use for your experiment, you can move on to making the other decisions you need to like selecting the right reagents and detection method – TaqMan or SYBR Green. To help with this decision we have an Ask TaqMan video dedicated to qPCR reagent selection called ‘Choosing the Right Master Mix.’
We also make it easy to find the best reagents for your chosen RT and qPCR detection method with a handy online selection guide, available on the thermofisher.com website.
So whether you are doing gene expression analysis or RNA virus detection make sure to choose the Reverse transcription method that’s best for you and your experiment. It can make a world of difference.
If you have more questions on RT methods or any other qPCR questions, remember to Ask TaqMan and submit your questions on our website www.thermofisher.com/ask
Thanks for watching!