RNA interference (RNAi) is a process of post-transcriptional gene silencing medi
ID: 203632 • Letter: R
Question
RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by short double-stranded RNA molecules called siRNA (small interfering RNAs). In mammalian cells, transfection of 21–22 nucleotide siRNAs leads to degradation of mRNA molecules that contain the same sequence as the siRNA. In the following experiment, siRNA and knockout mice are used to investigate two related cell surface proteins designated p24 and p25 that are suspected to be cellular receptors for the uptake of a newly isolated virus.
A. To test the efficacy of RNAi in cells, siRNAs specific to cell surface proteins p24 (siRNA–p24) and p25 (siRNA–p25) are transfected individually into cultured mouse cells. RNA is extracted from these transfected cells and the mRNA for proteins p24 and p25 are detected on Northern blots using labeled p24 cDNA or p25 cDNA as probes. The control for this experiment is a mock transfection with no siRNA. What do you conclude from this Northern blot about the specificity of the siRNAs for their target mRNAs?
Probe p24 cDNA Probe p25 cDNA Control SiRNA-p24 siRNA-p25 Control siRNA-p24 siRNA-p25Explanation / Answer
Knock out mice is the genetically modified mice, in which existing gene is inactivated of knock out. Here we are used know out mice for the gene for protein p24 and p25 surface receptor. Which means our mice do not have p24 and p25 gene. When the mice is transfected with si-RNA p24 and si-RNA p25, no RNA sequences were detected because the genes are already deactivated hence mRNA will not form and si-RNA for p24 and p25 will not work.
Because of this as shown in figure when we performed nouthern blot for p24 probe and p25 probe. in case of probe p24 no mRNA present for binding cDNA (p24 gene is know out) hence the lighter band is observed as compared to control and p25 cDNA probe (here p25 is normal so dark band is observed). vice verse. In figure2, for probe p25 cDNA probe, no mRNA sequences are present for p25 cDNA probe for hybridization hence siRNA –p25 has lighter band than control and p24 probe (here p24 is normal) (because as discussed no mRNA is present for si-RNA to work in both case).
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