Is it a new designed primer or your primer refers to the publication? If you are using a new designed primer, you can do that to be sure that your primer design is good but if you follow the publication, the optimized condition will be provided. Hope this help.

Yes, use exactly the same conditions that you would for the real-time PCR and run on a 2% agarose gel. You can also do this when you have run your qPCR if you are using probes (you can do a melt curve analysis if you are using SYBR), to make sure that you have product. I agree with the previous answer in making sure that you use either the published conditions, or the conditions that are recommended by the kit provider.

Yes, always test out your primers with regular PCR before you start PCR. This will help you confirm that you are getting the expected size product and determine the optimum annealing temperature. It's much cheaper to test this out with regular PCR than the expensive qPCR reagents. Good luck!

Running a 2% agarose gel is an good qualitative method that can help determine the optimal annealing temperature and expected product size, but should not be used for publication. It is also less costly than other PCR methods. For relative PCR data to be meaningful, the PCR reaction must be terminated when the products from both the internal control and the gene of interest are detectable.

Depending on your specific application, I would definitely recommend looking at the different types of PCR. Reverse transcriptase PCR (RT-PCR) has its benefits because it is highly sensitive. Primers are extremely important! You need stem-loop primers for miRNA. Otherwise, using mRNA as the source of the target DNA may result in the removal of the introns (long introns can interrupt the coding segments, but may be what you are interested in). It is important to note that RNA is less stable since it is single stranded. PCR using extracted DNA will not contain the regions you are interested in.

So your next question should be your detection method for quantitative PCR. There are two types of detection methods for RT-qPCR, namely dye-based detection and probe-based detection. Probe-based is more specific, yet more costly.

http://www.enzolifesciences.com/science-center/technotes/2017/march/what-are-the-differences-between-pcr-rt-pcr-qpcr-and-rt-qpcr?/?utm_source=2017-03-31-TN&utm_medium=email&utm_campaign=TechNotes

https://www.neb.com/faqs/2016/11/14/should-i-use-probe-or-dye-detection-for-my-qpcr-assays

yes, it is recommended to perform cPCR before TR-PCR, even use few samples from each group to confirm your results, also to exclude any unwanted results that may be not due to amplification and primers.

thanks

As a first check of your primers, you may choose for a cheaper PCR variant such as conventional PCR. Size detemination is not adequate for confimation of amplicons' identity. That sequencing. However, as first try out, your conventional PCR is not optimized and a band in agarose gel can be contaminated with by products.

So I recommend to a quick start in qPVR with EvaGreen as intercalating dye, even if you want to develeop a test with a hydrolysis probe. First tnings to do are:

1) design more than 1 primerpair if possible; check primers at about 15 criteria

2) if you want, a quick scan in conventional PCR

3) test your primersets at 250nm at 5 oC below Tm in qPCR and perform a melting curve. Use so many cycles that a plateau is reached for about 10 cycles. Analyse amplification curves with flatform software, but preferable with non-baseline corrected amplification curves and LinRegPCR. Check for by product with melting curve analysis MCA

4) choose 2-3 best primerpairs and perform gradient PCR followed by melting curves (see 3)

5) choose the best Ta and perform a primer titration, from 62,5 nm, 125, 250, 500 nm in a checkerboard, foolewed by a melting curve, analyse according to 3)

6) choose the best primer concentration and make a 10X dilution series of you target with possible 8-10 logs at optimal Ta, including a dilution that should not contain any target. Perform qPCR and melting curve analysis. Select those target dilutions that have equidistant Cqs and contain no by products to establish the AMR (analytical measurement range)

7) etcera

Well in terms of product size, it wouldn't be much of a help, since you would know the size initially when you designed the primers. However, for the optimization of the annealing temperature , it is recommended that you do the conventional PCR first and confirm the amplification as well. Similarly, since you will be doing reverse transcription first, it is possible to perform a semi - quantitative analysis as in using imaging softwares like ImageJ to see whether or not there is significant difference between the experimental and the control sample. For this, simply perform the conventional PCR for the miRNA as well as reference gene from both sample and control cells and then do the gel run at the same time for both of the samples, take the picture and analyze them using the software.Although this no way can give an accurate result as that of real time PCR but still should give you a preliminary idea at least.

Hi Noha,

It is certainly cheaper to do a regular PCR test before RT-PCR, but I've found that optimizing your conditions there may not be effective unless you are using the same master mix. I use a pre-made master mix with SYBR for RT-PCR, and the component concentrations (MgCl2, dNTPs etc) aren't known so I find it more accurate to just validate with that master mix rather than with a different master mix with different component concentrations, which can change your results dramatically.

Hope that helps!

Renae

Yes. It is a good thing to make sure about your desired amplicon size and also to check the primer is working perfectly. By going through the conventional PCR once you will be sure about all these.

Yes, always conventional PCR will help you to getting the size of amplicon and optimizing annealing temperature of the new design primer. The results of conventional PCR give you idea and assurance of the best qPCR result. Good luck!

I advise AGAINST optimising annealing temperature with conventional PCR if you want to develop a qPCR assay

Checking the product size is in principle a sound quality check, but it comes at a cost: you have to handle your product in the open lab. This undermines one of the most important advantages of real time PCR: the fact that the amplified sample is never opened and cannot, therefore, contaminate the lab.

So, if you are going to do gel electrophoresis on your PCR product, make sure you do it in a dedicate post-PCR lab with a well established and controlled one-way flow. Decontaminate stringently with hypochlorite and/or UV afterwards. Note that the gel buffer and everything that has been in contact with it are contaminated.

That's why I avoid it. The experiment is simple. Decontaminating afterwards is a major operation.