When real-time polymerase chain reaction came out during the 90s the words kinetic PCR analysis or real-time PCR were most commonly used [1][2][3]. Also, real-time quantitative RT-PCR was used to indicate RT-qPCR [4]. When real-time PCR became more common in the 2000s it was more often shortened to RT-PCR or qPCR (quantitative PCR) [5][6][7], because of its main function to quantify genomic fragments in real-time. It certainly makes sense to abbreviate real-time PCR into RT-PCR and all would be well if there already hadn't been reverse transcription PCR [8]. Yet it was and we are all thankful for it, except a little confused about the terminology.
So when you are reading about RT-PCR, how do you know you are reading about the right PCR? Well, you have to pay attention to the details. Also note that after the devising of MIQE guidelines in 2009 [9], there have been fewer mix-ups between the methods.
We have already discussed qPCR in our previous blog post. It’s a quantitative method used to detect, quantify and characterize genomic fragments in real-time. Reverse transcription PCR, which will be called RT-PCR in the following text and blog posts, is a method that combines reverse transcription with PCR to detect RNA transcripts. Therefore while the abbreviations may be the same, the methods are quite different. Then there is also a third option called RT-qPCR that combines both methods and is used to quantify gene expression.
One-step or two-step reaction
Now that you have gotten this far you might be a little confused about the one-step/ two-step reaction. RT-PCR and RT-qPCR obviously have two parts, so how can those be done in a single reaction or why is there even a need for two options?
With a two-step reaction, first, you perform the reverse transcription reaction in one tube and then the PCR reaction in another tube - basically, the two steps can be considered different experiments. You can synthesize cDNA one day and then perform PCR with it in the next day or next week or whenever you want. That way you can stock cDNA and use it for more than one experiment. A two-step approach is also good when there is a limited amount of starting material, when you want to optimize the steps separately or when you want to detect multiple targets from the same sample.
With a one-step reaction, the whole experiment will be done in a single tube, where reverse transcription and PCR components are all mixed together, which is obviously much easier and faster than doing the steps separately. There is also a much smaller possibility of contamination and with excellent products from Solis BioDyne, you can also use one-step RT-qPCR to detect multiple targets from the same sample.
All that is left now is to check out our product list and choose the product most compatible with your needs.
Be sure to read our following blog posts to find out more about different PCR variations and which product might suit you the best.
Citations:
[1] Higuchi, R., Fockler, C., Dollinger, G. et al. Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions. Nat Biotechnol 11, 1026–1030 (1993). https://doi.org/10.1038/nbt0993-1026
[2] Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996 Oct;6(10):986-94. doi: 10.1101/gr.6.10.986. PMID: 8908518.
[3] Bustin, S. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays, Journal of Molecular Endocrinology, 25(2), 169-193. Retrieved Aug 2, 2021, from https://jme.bioscientifica.com/view/journals/jme/25/2/169.xml
[4] Fink, L., Seeger, W., Ermert, L. et al. Real-time quantitative RT–PCR after laser-assisted cell picking.Nat Med 4, 1329–1333 (1998). https://doi.org/10.1038/3327
[5] Deepak, S., Kottapalli, K., Rakwal, R., Oros, G., Rangappa, K., Iwahashi, H., Masuo, Y., & Agrawal, G. (2007). Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes. Current genomics, 8(4), 234–251. https://doi.org/10.2174/138920207781386960
[6] Oliveira, A. C., Vallim, M. A., Semighini, C. P., Araújo, W. L., Goldman, G. H., & Machado, M. A. (2002). Quantification of Xylella fastidiosa from Citrus Trees by Real-Time Polymerase Chain Reaction Assay. Phytopathology, 92(10), 1048–1054. doi:10.1094/phyto.2002.92.10.1048
[7] Herrmann, B., Cavaglia Larsson, V., Rubin, C.-J., Sund, F., Eriksson, B.-M., Arvidson, J., … Blomberg, J. (2004). Comparison of a Duplex Quantitative Real-Time PCR Assay and the COBAS Amplicor CMV Monitor Test for Detection of Cytomegalovirus. Journal of Clinical Microbiology, 42(5), 1909–1914.doi:10.1128/jcm.42.5.1909-1914.2004
[8] Mocharla, H., Mocharia, R., & Hodes, M. (1990). Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping. Gene, 93(2), 271–275. doi:10.1016/0378-1119(90)90235-j
[9] Stephen A Bustin, Vladimir Benes, Jeremy A Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W Pfaffl, Gregory L Shipley, Jo Vandesompele, Carl T Wittwer, The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry, Volume 55, Issue 4, 1 April 2009, Pages 611–622, https://doi.org/10.1373/clinchem.2008.112797
Lyophilization, also known as freeze-drying, is in simple terms a water-removal process that increases product stability and preserves its functionality. Our new SolisFAST® Lyo-Ready qPCR Kit with UNG represents an optimized lyophilization-compatible qPCR solution to enhance the simplicity, convenience, and speed of diagnostic and applied testing.
The running joke with PCR is that if something can go wrong, it will go wrong. Quite often it’s even impossible to determine why some samples turned out fine while the others did not. In a situation like this, it would be amazing to know some trick or a secret to avoid spending all the time and resources to do the experiment again. Here are a few we are willing to share so that you could find love for PCR.
In research, every day different methods are used to discover something new, whether it is a new disease, medicine, or something else. Often these methods were developed long ago and are confirmed to be doing what they are supposed to do. However, as technology develops so do new methods. This is exactly what Professor Steven Williams’ lab is doing at Smith College – developing new methods to be used in research and diagnostics.
As an alternative to PCR, the loop-mediated isothermal amplification (LAMP) reaction has been developed for DNA detection. The LAMP test is fast, simple, and sensitive.
