Hi everyone.
I'm trying to knock-down COUP-TFII in K562 cells using short-hairpins.
I'm using viral transduction.
I have 5 construct and on WB (day 4 after transdcution) it looks goed. Also the amount of the protein is the same if I look at the control (nucleophosmin). But I've just performed the qPCR and it's a disaster. sh 3 only increase the amount of COUP.
The only thing take to concideration is that for qPCR I've used control with gpf plasmid to normalise and on western blot I've used the wild type to do so. But it shouldnt be the problem.
Thank you in advance!
It is possible that the short hairpin RNA (shRNA) constructs are not efficiently reducing the expression of COUP-TFII at the mRNA level, as indicated by the qPCR results. There could be a number of reasons for this.
One possibility is that the shRNAs are not being effectively expressed or processed in the cells. It is important to ensure that the shRNA constructs are being transduced effectively into the cells and that the shRNAs are being expressed and processed correctly. You could try using a fluorescently labeled shRNA construct or a reporter gene, such as GFP, to monitor the expression and processing of the shRNA in the cells.
Another possibility is that the shRNA constructs are not targeting the COUP-TFII mRNA effectively. It is important to ensure that the shRNA sequence is complementary to the COUP-TFII mRNA and that the shRNA is being expressed at a high enough level to effectively reduce the expression of COUP-TFII. You could try using different shRNA constructs with different target sequences to see if they are more effective at reducing COUP-TFII expression.
It is also possible that the COUP-TFII protein is being stabilized and is not being degraded at a normal rate. This could explain why the protein levels are not decreasing even though the mRNA levels are decreasing. In this case, you may need to use additional approaches to target the COUP-TFII protein directly, such as using small molecule inhibitors or CRISPR/Cas9 gene editing.
It is also possible that there are other factors at play that are influencing the expression of COUP-TFII in your cells. It is important to carefully consider all of the variables in your experimental setup and try to identify any potential sources of variability or confounding factors.
Hi
I also agree wit the suggestions above. The expression of the shRNA should be ascertained int the lines vis-a-vis the level of expression of the target gene . If the shRNA levels are on lower side you may need to address it. Also, this aspect may have been addressed in the beginning that it is better to design the qPCR primers for validation of target mRNAs in the region flanking the target site. This provides an estimate of known-down due to cleavage. In addition, the target gene in the control cell lines should have been taken to compare the level of down-regulation instead of the plasmid..
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