week 1 section C

Why do we start with harvested bacterial cells when measuring FIX expression?

Because the FIX mRNA is inside the bacteria, and expression only occurs in cells that have been induced with IPTG. Harvesting them lets us analyse the actual gene activity.

Why is RNA extracted rather than DNA for qPCR expression analysis?

RNA reflects active gene expression — it's transcribed from the FIX gene. DNA is constant in all cells, so it can’t show differences in expression levels.

Why is reverse transcription used to make cDNA from RNA?

Because qPCR enzymes can’t work with RNA directly. Converting to cDNA allows accurate and efficient amplification and quantification of gene transcripts.

What is the advantage of using qPCR over standard PCR for expression analysis?

qPCR allows real-time quantification of gene expression levels, not just presence/absence. It shows how much FIX mRNA is in the sample based on fluorescence.

Why do we normalise FIX expression to a housekeeping gene in qPCR?

To control for variability in RNA amounts or quality between samples. Housekeeping genes have stable expression, providing a baseline to compare FIX levels.

What’s the benefit of using the ΔΔCt method in qPCR data analysis?

It allows relative comparison of FIX expression across conditions (e.g., induced vs. uninduced), making it easy to quantify fold-changes in expression.