I want to determine the amount of expression by performing qPCR of a bacterial gene region. For this, I compared some primers found in the literature. Some of these primers targeted the starting part of the gene, while some expressed the last part of the gene. Does it matter where the primer is linked to the gene to determine the expression level by making qpcr? When making qpcr, does the binding of the primer from the end or beginning to the gene site affect the expression level?
Hello Berfin,
The region of the gene shouldn't matter, because you are working with cDNA synthesized from the RNA of the whole gene that was synthesized by the cells themselves before this RNA was extracted.
What should be considered is, if these primers are going to make secondary structures that would hinder the qPCR reaction, if they are going to "stick" together forming self- and cross-dimers (which, again, might decrease the reaction efficiency), what is the length of the product that would be synthesized with the use of these primers, if their annealing temperatures are similar, etc. Here is a link to a very useful online program that can help you check if your primers are good:
https://eurofinsgenomics.eu/en/ecom/tools/oligo-analysis.aspx
Then, the efficiency of the qPCR with the chosen primers should be checked, because even those primers that seem perfect in theory may have low efficiency in the actual reaction.
Hope this helps!
Best wishes,
Dina Malkeyeva
Berfin Eroğlu
1. If your gene has multiple functional transcripts you need to locate primers in the region that is common for them.
2. If your gene is expressed at low level, it maybe be better to locate them closer to 5` end, because cDNA synthesised with R6 oligonucleotides is enriched with 5` molecules.
I would recommend testing several primer pairs in different regions of the transcript, in order to make sure that the PCR is working.
Note that DNA sequences are only perfectly linear and complete on paper, in reality, you can always have secondary stucture slowing down or even inhibiting amplification (e.g. GC-rich or selfcomplementary regions). Primer pairs at the 3'-end of transcripts might be better in case that the reverse transcription to cDNA was not complete.
It is likely that you will see some differences using different primer pairs on the same transcript.
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