I am doing viability assay after gene overexpression. For overexpression, I used AAV vector and checked the gene expression using RTqPCR. Although, it was statistically significant compared to the control, there was a considerable variation in gene expression between samples. What could be the reason for this variation? Does this variation could have an effect on the results of other experiments such as viability assay? If so, how could I confirm a stable gene expression? Thank you.
Rakesh Sharma
Transduction Efficiency:Variation in the efficiency of AAV vector delivery can lead to different levels of gene expression across samples. Vector Copy Number:Differences in the number of vector copies integrated into the host genome can cause variability in expression. Cellular Context:Differences in the cellular environment or the state of the cells can impact gene expression levels. Use of Stable Cell Lines:Generate stable cell lines with integrated vectors to reduce variability in gene expression. Clonal Selection:Select and expand clones with consistent expression levels. Normalization:Normalize gene expression levels to a housekeeping gene or another internal control to account for sample-to-sample variation. Single-Cell Analysis:Perform single-cell RNA-Seq or flow cytometry to analyze expression levels at the single-cell level and confirm uniform expression. Multiple Replicates:Use biological and technical replicates to account for variability and ensure reproducibility.
Reasons for Variation in Gene Expression
Confirming Stable Gene Expression
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