Hello every one i want to know about primers for qRT-PCR and RNAi primers. I make dsRNA from ORF region of gene sequence and inject into pest at different dose. Now my question is that whether i design primer inside ORF region for qRT-PCR to check knock down dose wise of gene or out side ORF region?
rt-qPCR analysis provides a convenient and reliable method
for evaluating knockdown effciency and functional effects of
RNAi-mediated gene silencing, as demonstrated by the use
of this tool for monitoring knockdown of MYCN and TP53
genes, which are pivotal in neuroblastoma pathogenesis.
Successful application of rt-qPCR requires careful attention to
all of the steps in the assay workflow, including primer design
and evaluation, template preparation, normalization strategy,
and data analysis.
Respected Ali and Mehdi thanks for your quick and positive responce actually i am going to knock down cuticle gene with injected dsRNA trough RNAi, I clone new genes from insect skin and then make dsRNA of that gene ORF region with Promoter primers. All gene have only one ORF region about 585bp. my question was that whether i design primer inside same ORF for qRT-PCR to check gene silencing or out side in UTR region.
Hi Saad
your question seems bit confusing, i barely understand a little.
yes, you can design qRT-PCR and RNAi primers from ORF Region of your gene of interest.
Hi Saad,
if you are doing systemic (parental) RNAi I would not mind about sequence overlay between dsRNA and qPCR fragment, as the mother should only deliver fully processed dsRNA pieces to the offspring. If you inject embryos or adults there is a small chance that your RNA extraction may pick up some of the injected RNA. However the dsRNA should be cut by the RNAi machinery into pieces to small for PCR or, if not processed, should be still double-stranded so that the cDNA synthesis does not work on it. However, if you have some spare space in your gene(s) it is always saver (and better to write in publications) to design the qPCR primers outside of your dsRNA. In my experience around 300bp are enough for efficient knockdown by dsRNAi. This would leave you around 180 bp to design qPCR primers on.
Hope, that helped.
I designed the qPCR primers outside dsRNA region preferably towards the 5' regions and it worked excellent.
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