In the experiments shown siRNAs that were specific to ADAR 1 and ADAR2 were sepa
ID: 162046 • Letter: I
Question
In the experiments shown siRNAs that were specific to ADAR 1 and ADAR2 were separately transferred into a human liver cell line, and the effects analyzed in various ways. Figures A and B show the effects of the siRNAs on the ADAR1 and ADAR2 proteins. What is being measured in this experiment? Do these results show what we would expect? Figures C and D show the effects of the ADAR siRNAs on the AhR (Aryl hydrocarbon receptor) RNA and protein. Please state what you see in the Figures. What techniques were used to obtain these results? How would you interpret what you see in terms of the known roles of the ADAR proteins on RNA? How can you explain the results seen in Fig D vs C?Explanation / Answer
Figure A.
siRNA against ADAR1 and ADAR2 were transfected on liver cells. Efficiency of transfection was measured by checking the expression level of ADAR1 by western blot. ADAR1 siRNA drastically decrease the level of ADAR1 but ADAR2 siRNA was unable to do the same. Graph showing the comparative level of ADAR1 with respect to GAPDH which is used for normalization
Figure B
siRNA against ADAR1 and ADAR2 were transfected on liver cells. Efficiency of transfection was measured by checking the expression level of ADAR2 by western blot. ADAR1 siRNA drastically decrease the level of ADAR2 but ADAR1 siRNA was unable to do the same. Graph showing the comparative level of ADAR1 with respect to GAPDH which is used for normalization.
We were expecting that ADAR1 siRNA will silence ADAR1 gene and ADAR2 siRNA will silence ADAR2 gene. We got the result that we were expecting.
Figure C
siRNA against ADAR1 and ADAR2 were transfected on liver cells to check the effect of gene silencing of ADAR1 and ADAR2 on AhR mRNA level. ADAR1 and ADAR2 silencing doesn’t affect the level of mRNA of AhR gene. Graph showing the comparative level of AhR mRNA with respect to GAPDH mRNA, which is used as RT-PCR control.
RT-PCR was used for the experiment and GAPDH is used for normalization.
Figure D
siRNA against ADAR1 and ADAR2 were transfected on liver cells to check the effect of gene silencing of ADAR1 and ADAR2 on AhR protein level. ADAR1 silencing increased the level of AhR protein by two-fold whereas ADAR2 silencing doesn’t affect the expression of AhR protein. Graph showing the comparative level of AhR expression level with respect to GAPDH which is used for normalization
By comparing figure 3 and 4 we can interpret that ADAR1 regulates AhR gene negatively in a post-transcriptional manner