The question provides most of the details. Here is a little information as I have tried to troubleshoot.
The gene introduced is occludin GFP in HepG2 cells via a lentiviral system.
The plasmids are sequence validated.
I have primers that span exon/exon boundaries, have DNAse I treated the RNA and there is no DNA contamination of the primers.
Reference gene between control and overexpressed have identical ct values.
I'm assuming you mean qPCR of your RNA. Check your product sizes on an agarose gel to make sure you don't have genomic DNA contamination. DNase doesn't always go to completion. Also, make sure your primers are specific and not amplifying a related gene. What were your Ct values of your control gene in each sample? Otherwise similar Ct values could be due to different amounts of starting material.
Not sure what you mean by "overexpressed".
If the encoded protein is expressed, the gene must be transcribed (and translated), but does it really matter at what level transcription takes place?
Maybe a description of your experimental objectives would help?
Does it really matter? Your objective is to express the protein and you demonstrated that. Isn't it?. You have some technical problem with your RNA detection that will take time to solve. My advice is that you focus on your research goal and move forward.
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