I have constructed a gene knockout in Candida albicans via the introduction of a premature stop codon in the ORF. I have repeatedly shown that the respective protein is no longer being expressed in a Yeast-Carbon Base + 1%BSA growth assay (the protein acts as a proteinase and without it, it cannot break down the BSA and use it as a growth source). When I use RT-qPCR to confirm the lack of gene expression, however, it shows expression levels comparable to WT. I am using primers downstream of the stop codon, so cannot understand why I am still seeing expression. Any help would be very much appreciated! Thanks
Hi Olivia,
Introducing a stop codon in the gene sequence will not suppress the gene's transcription, but will prematurely end the mRNA's translation. Therefore, it is normal to detect the transcript using RT-qPCR, whereas the protein is expected to not be expressed anymore in your knockout cells.
Hoping that helps,
Jérôme
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