We attempted to downregulate the expression of a specific gene using lentiviral shRNA, but instead observed overexpression. What factors should I investigate to determine the cause of this unexpected result?
We used the pLK0.1 shRNA plasmid, psPAX2 packaging plasmid, and pMD2.G envelope plasmid, followed by 7 days of puro selection. When we compared the expression levels of the 5 shRNAs to that of the scramble shRNA, we found that they were similar or increased by 50%. Can you please let me know if you have experienced a similar case?
Check your qPCR primers do not bind to your shRNA plasmid or the shRNA itself.
It is not uncommon to observe unexpected results when attempting to downregulate gene expression using shRNA. Here are some factors that you should investigate to determine the cause of the unexpected overexpression result:
In addition to the above factors, it is also important to rule out any technical errors that may have occurred during the experimental procedure. If none of these factors explain the unexpected result, it may be worthwhile to consider alternative methods for gene knockdown, such as CRISPR-Cas9.
Robert Adolf Brinzer , Mamlikatu Ilmi Azizah Thank you for the detailed explanation~!!
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