I need to compare between different treatments through real-time PCR technique upon Liriomyza trifolli (insect pest) genome, but I could not know what is the sequence of primers shall I use to detect the activity of enzymes related to oxidative stress my treatments can do upon this insect gene expression. can you help me ?!
"How to design primers for genes that have not been studied in the literature - and don't even have gene entry information in the PubMed databases?"
Apparently none of these genes have been characterized in your model of choice. Zero hits in PubMed.
What you can do is to to go to the PubMed “Gene” database, pick several model species of different phylogenetic distance to your target species. Do include the fruit fly (Drosophila m.) and at least two more insects.
https://www.ncbi.nlm.nih.gov/gene?term=sod[gene]%20AND%20alive[prop]%20NOT%20newentry[gene]%20&sort=weight
Download their DNA sequences (coding sequences, CDS) for the genes, one by one (FASTA format).
Then, for each gene, run a multiple alignment analysis. (There are many softwares, ask a friend or labmate to help).
https://www.genome.jp/tools-bin/clustalw
https://www.ebi.ac.uk/Tools/msa/clustalw2/
Wiki: https://en.wikipedia.org/wiki/Multiple_sequence_alignment
When you have the multiple alignment results, say for "Cu,Zn SOD", then you will see which regions of the gene are highly conserved (little difference across all species). Pick these conserved regions to design your new primers (again, many softwares for assisting primer design. Don't worry, most are very easy, as long as you are guided, and given help).
For real-time PCR, the amplicon (that is, the DNA product you get from PCR reaction) should contain about 300 - 350 base-pairs. This length is near optimal, not too long (so that the amplification efficiency is low), and not too short (so that it compromises your selectivity in gene detection).
Primer design principles: https://www.addgene.org/protocols/primer-design/
http://cshprotocols.cshlp.org/content/2009/3/pdb.ip65.abstract
You should design several pairs of primers (forward versus reverse) and try out what's best for your experiments.
Good luck.
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