Question

3. EML is a protein that is produced at low levels in healthy cells, but is highly expressed in cancer cells. You know that EML expression correlates with HER2 activity in cancer cells, but you do not know if EML causes HER2 activation or if HER2 activation induces EML expression.

a. Your PI thinks that it would be a good idea to look into quantifying expression of EML. What method would you use?

b. The particular method you most likely selected in part a utilizes SYBR green and generates a Ct value. What does SYBR green do? What is the point of having a Ct value? After using CRISPR/Cas9 to knock out the HER2 gene in a healthy cell line, you transform this CRISPR-edited HER2-/- cell line into cancer cells. When comparing the expression of EML in the HER2-/- vs the wild type cancer (assume the HER2 gene is intact) cell lines, you find that the Ct value of EML is significantly higher in the HER2-/- cells. What conclusion about the relationship between HER2 and EML does this experiment suggest?

Answer 1

3.a. To quantify the expression of EML we can use several methods like western blotting, ELISA, RT-PCR to quantify the expression of a protein. to check the expression relation between EML and HER2, either one of them can be inhibited using either siRNA technology, specific inhibitors or knocking out genes using CRISPR/Cas9.

b. particular method selected, utilizes SYBR green to bind to DNA, generating Ct (cycle threshold) value is PCR. In PCR, when DNA concentration increases SYBR attachment to DNA increases and more flouroscence is observed. on comparison of EML expression as said above, Ct value were high in HER2 knockout cells, which establishes negative relation between HER2 and EML. If activation of either of them were to be regulated by the other one then, the expression would have down-regulated.