Iam searching for Catalase primer sequence for dromadrey camel, when I search in NCBI, I found one and make pick primers to found the sequence. I get 10 primer pair
Iam asked about which one is better for gene expression analysis!
This is the link: https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi?ctg_time=1631749594&job_key=lpxJJRXVGH0_R4hChSKscP85vULSKqZf0w
Dear Fatma
First, you need to choose the methods regarding the expression analysis. I assume you will use qPCR method for expression analysis. The primer design will be different when you use qPCR probe vs. intercalating dye such as SYBR green.
If you will use qPCR probes, the primer should be optimized and designed together with the probe. If you use intercalating dye like SYBR, the recommended PCR product should be around 70-200 bp, and I recommended to order more than 1 set of primer to compare its efficiency later.
More important thing, primer and probe designs should be screened for self-dimers, heterodimers against the 2 primers, as well as hairpins. The ΔG value of any self-dimers, hairpins, and heterodimers should be weaker (more positive) than –9.0 kcal/mole. Positive numbers indicate that the actual secondary structure shown will not form at all. Use the free, IDT, online OligoAnalyzer Tool for this purpose or other available program online.
From the amplicon size, specificity and Tm primer Pair no 7,8 and 9 looks good. Further secondary structure screening on those pairs is recommended. And It is also crucial to reflect monovalent cation, Mg2+, and dNTPs concentration in the Tm calculation to check the potential secondary structure
Good luck.
Hello Fatma B. Aboelwafa, ideally it is better to test all the set of 10 pairs (or choose a few among them with given criteria: melting temperature, self hybridization, hairpins...) and see which pair gives you a good PCR product. This product should be sequenced to see if it is specific to the target "dromedary catalase" by blasting it.
Primers are cheap - order them all. The "best" pair will be the ones that give 95-105% efficiency in your qPCR assay.
That being said, make sure that your primers will anneal to cDNA from your gene. It's better to have at least one of the primers span across 2 exons to avoid amplifying gDNA.
In my experience, primers that give one nice bright band across a range of annealing temps are generally the best in qPCR (when you are restricted to using the annealing temp specified in your enzyme mix).
Good luck!
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