hello everyone, i hope i can get help on this.
so i knockdown a gene using RNAi and i see its knockdown phenotype on the plate, however when i extract the RNA and reach qPCR i see upregulation in most of the replicate. does anyone know why or if im doing something wrong? I already troubleshooted my SYBR green and cDNA synthesis Kit and they’re working perfectly fine, and my RNA quality and purity are also great.
Hello Maryam ..
The best way to confirm successful knockdown of a target gene by siRNA is to perform a Western Blot. However, since you have tried RT-qPCR, which is no doubt an alternative approach, designing of primers for RT-qPCR experiment should be considered important, as using a primer set targeted to the wrong region may give you false negative results.
You need to check the primers for RT-qPCR as this could be one of the reasons. You may want to refer to the article attached below for some useful information on detection of siRNA induced mRNA silencing by RT-qPCR.
Article Detection of siRNA induced mRNA silencing by RT-qPCR: Consid...
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