Hi Giorgia,

I suppose it comes down to what you want from the RT-qPCR. A one step RT-qPCR will allow you to process a larger number of samples, as it will be less hands-on, and will also reduce the chances of contamination or sample mix up. A two step will allow you greater flexibility in the amount of RT product added to the qPCR and flexibility in the priming you use for the RT step (oligo dT, randomers or gene specific priming).

From a personal point of view I preferred a one step RT-qPCR for the plant virus diagnostics I used to do as this reduced the handling steps and the chances of sample mix up or cross contamination.

So it really is up to you.

Happy PCRing 

Hi Giorgia,

I suppose it comes down to what you want from the RT-qPCR. A one step RT-qPCR will allow you to process a larger number of samples, as it will be less hands-on, and will also reduce the chances of contamination or sample mix up. A two step will allow you greater flexibility in the amount of RT product added to the qPCR and flexibility in the priming you use for the RT step (oligo dT, randomers or gene specific priming).

From a personal point of view I preferred a one step RT-qPCR for the plant virus diagnostics I used to do as this reduced the handling steps and the chances of sample mix up or cross contamination.

So it really is up to you.

Happy PCRing 

Thanks for your point of view.

• agreed
• On balance, like Chris, I think they are largely interchangeable unless you are dealing with known low copy number genes; in which case 2 step can be optimised and made more sensitive
• The only clear cut general preference I have, based on personal experience, is if you are dealing with small amounts of starting material (< 10^(5) cells); in which case combined RNA extraction and cDNA synthesis is without doubt better
• But in terms of cDNA synthesis and especially PCR it is a matter of personal choice, available kits aptitude and experience etc

Hi Giorgia,

 The advantages of One-step RT-qPCR are : 

-Accurate representation of target copy number

-Simple and rapid

-Fewer pipetting steps (reducing possible errors and contamination)

-Best option for high-throughput screening

-Best method when only a few assays are run repeatedly

-Multiplex PCR of gene of interest and control can be done in single well, from same RNA sample

The Considerations:

-Usually less sensitive as it is impossible to optimize the two reactions separately

-Difficult to troubleshoot RT step

- No stock of cDNA

While in 2- step

The advantages are :

-Two buffers optimized for independent RT and real-time PCR

-Highly sensitive

-Potentially more efficient because random primers and oligo d(T) can be used

-Possibility to stock cDNA to quantify several targets

-Recommended when the reaction is performed with a limiting amount of starting material.

The considerations are :

-Time consuming

-More pipetting steps (increases possible error and contamination)

-Requires more optimization

Best regards 

I thank all of you for responding to my question

For the standard in the RT-qPCR is better I use cDNA o cRNA?

• For most genes and abundant genes definitely cDNA
• However for definite low copy number genes cDNA will work but cRNA might be better 

Hi Giorgia, these following links have enough information to understand about one step and two step RT qPCR in quite simple words.

https://www.thermofisher.com/sa/en/home/life-science/pcr/reverse-transcription/rt-pcr/one-two-step-rt-pcr.html

http://www.bioline.com/sg/one-step-vs-two-step-real-time-pcr.html

http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2011/09/12/one-step-two-step

But I would still vote for two-step RT qPCR, as more work is really worthy if I managed to achieve the desired results with proper knowing and troubleshooting every steps carefully till the generation of my proper results. Contamination issues are mainly dependent on your personal skills and techniques, and you can overcome those.

In one step case, you must have to check the RNA quality or integrity by all means before applying that RNA sample into an one-step system. So at least, later you will be on a safer side if something unexplained occurs in your reaction, if you are not sure about your RNA quality from the beginning so how you would know what actually has failed you. And never forget, my RNA which I have extracted is very precious as I got it by painful procedures and often we don't get it in a suitable amount and concentration, so just for small assays, one-step approach may be good but for comprehensive large-scale studies two-step is probably the right choice.

As we also know that the extracted RNA may contain  variety of known inhibitors, which can inhibit RT step or qPCR step or both, occurring together in the same reaction tube in one-step case, so to avoid this,  the two-step is still a better choice and this really matters. In one step, we usually use GSP primers. In total RNA, the concentration of different genes are not known to us and we cant do anything about it as we have one type of specific primer here to look only at specified or certain target.

One-step now is considered more sensitive, as it can detect up to 0.01 pg of RNA, but for HKG here we usually need to perform separate reactions for each and every GOI of the assay, which is also labor intensive and costly. In two-step case, primer dimer formation is lesser than the one-step qPCR, as for one-step during the RT phase the lower temperatures nearly 40-50 C, may force our primers dedicated for qPCR phase to hybridize and generate PD. 

And in last by having your cDNA in stock in two-step case, you can repeat experiments if you want, or do alterations in concentrations and can get more flexibility than the one step case in terms of stable results, which we really need.

Anyway as per your situation, you are now at least in a better position to decide which type you want after this entire nice informative discussion.

Regards....