Hi Everybody
I have not done qPCR, and try to figure out the steps of that. From this regard, I am wondering Should primers designed for mRNA sequence or any other (cDNA, DNA, RNA) in qPCR, if mRNA, should it design for the whole sequence? or it can be designed for part of mRNA?
Also, I appreciate if anybody can provide me with a useful file for qPCR guides.
Thank you!
As for regular PCR, your primers should be designed based on the DNA sequence of your gene of interest, since the first step of qPCR is reverse transcription of the mRNA into DNA.
For qPCR, you want to design primers that will hybridize within your gene of interest to amplify approximately 175-200 bp. I suggest you input the whole gene sequence into primer3 (https://primer3plus.com/cgi-bin/dev/primer3plus.cgi), change the product size parameter to 150-250 bp and let it do the work for you.
As for a qPCR protocol, you may want to look for specific ones corresponding to the supplier you'll be using (SYBRGreen, iTaq etc.). Different protocols are available for different products, and that is also true for the RT (reverse transcription) step.
To expand on Martin Chenal 's answer, the primers should amplify a small (120-200 bp) region that is found in your mRNA. Ideally, the one primer should be "intron spanning" meaning that the primer will only anneal to spliced mature mRNA and not the gDNA. Also, you'll want the PCR product to cross an intron/exon boundary so that the product from your mRNA is shorter than a product from gDNA. That makes it very simple to determine if you have any residual gDNA in your reactions.
- Thank you so much, Katie A Burnette and Martin Chenal
- The other question that is worth asking is related to the first step for RNA preparation, do you usually prepare total RNA or mRNA? and do you add RNA stabilizer solution during RNA purification or try to keep it at a lower temperature as RNA is very sensitive.
Faezeh Jamechenarboo For the RNA prep I use a commercial kit with a spin column from Qiagen. I don't use additionnal stabilizers, but I do work exclusively on ice as soon as I elute the total RNA from the column. I also perform DNAse treatments to remove contaminant gDNA. You should also work in a RNAse-free environment (just wash your bench and pipettes with a specific spray that degrades RNAses).
Additional tip from me:
When running qRT-PCR with a newly designed primer pair, you should let the cycler perform a melting curve after your 40th cycle of amplification. Checking the melting curve data will give you information if your primers are specific and only generate one PCR product (as it should be).
Have a look on (Quantitative gene expression by real-time PCR: a complete protocol) byThomas D. Schmittgen. Very useful.
Firas Al-Tae , I could not find the real-time PCR: a complete protocol byThomas D. Schmittgen. Would you please send it to me if you have? Here's my email address:
Thank you so mcuh
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA