Question

You perform a real time qPCR experiment using mRNA isolated from HCT116 colon cancer cells grown in controlled atmosphere incubators at 0.5% or 16% O₂. You want to determine relative expression levels of the HIF1α response gene pyruvate dyhydrogenase kinase (PDHK). For sample one, the reaction takes 21 cycles to enter exponential phase and cross the cycle threshold. Sample 2 takes 26 cycles. Reactions amplifying β-tubulin as a loading control from samples 1 and 2 both took 22 cycles to cross the cycle threshold. What is the difference in PDHK mRNA concentration between samples 1 and 2? Your lab technician accidentally sprayed your sample tubes with ethanol, destroying your meticulous labeling (with sharpie). Which sample contains mRNA from the cells grown in 0.5% O₂? You may use the video tutorial if needed.

Answer 1

Ans:- PDHK expression in sample 1 is higher than sample 2. As PDHK in case of sample of 1 reach the threshold in just 21 cycle compared to sample 2 that hit the threshold at 26 cycle.

Now, the cycle difference between sample 1 and sample 2 is 5 cycles. Thus, roughly the concentration of mRNA is PDHK is 32 times higher in sample 1 than sample 2.

The true fold change is calculated if PCR efficiency is mentioned.

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