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