We checked qRT-PCR-based gene expression in our homozygous knockout cell line and mostly observed the downregulation of targets compared to wild-type control cells. We recently performed a transient knockdown using siRNAs against the same RBP in the wild-type control cell line, but the target genes were upregulated. What could be the reason behind these opposite effects in homozygous Knockout cell line vs transient knockdown in wild-type cells?
Kishor Gawade It is not uncommon to get such results in case of siRNA.
Pls refer to the following comparative study which presents a protocol to avoid false positive results.
Hahn P, Schmidt C, Weber M, Kang J, Bielke W. RNA interference: PCR strategies for the quantification of stable degradation-fragments derived from siRNA-targeted mRNAs. Biomol Eng. 2004 Nov;21(3-5):113-7. doi: 10.1016/j.bioeng.2004.09.001. PMID: 15567105.
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