You are analyzing the mechanism of action of particular short interfering RNAs.
ID: 74159 • Letter: Y
Question
You are analyzing the mechanism of action of particular short interfering RNAs. For your experiments, you transfect cells with a lucifierase reporter gene. You also transfect the cells with one of the following small RNAs: an unmatched siRNA, a perfect match RNA and a mismatch RNA. Figure A shows the luciferase activity in each sample, which is reflection of the ability of each small RNA to affect expression of luciferase protein. You then isolate mRNA and perform a Northern blot analysis, shown in Figure B, using a probe to the luciferase sequence. What conclusion can you make from these data? The mismatch silences by repressing transcription. The perfect match silences by cleavage of the mRNA. Neither the perfect match nor the mismatch affects luciferase protein expression. Both the perfect match and the mismatch silence by repressing ribosome elongation along the mRNA. The addition of the unmatched siRNA is required for expression of the luciferase reporter gene.Explanation / Answer
Ans- a)the mismatch silences by repressing transcription.
e)The addition of unmatched siRNA is required for the expression of luciferase reporter gene.
Luciferases make up a class of oxidative enzymes found in several species that enable the organisms that express them to “bioluminesce,” or emit light.
The luciferase reporter assay can be used to study gene expression as well as other cellular components and events that are involved in gene regulation. Its extreme sensitivity allows quantification of even small changes in transcription,
The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level
applications of luciferase reporters also include the study and effect of siRNA.RNA interference (RNAi) is a mechanism that inhibits gene expression before a gene is translated or by hindering transcription of specific genes. The two related classes of molecules that moderate RNAi processes are microRNA (miRNA) and small interfering RNA (siRNA), which are short noncoding RNAs.
The intensity of light emission is linearly related to the amount of luciferase and is measured using a luminometer. Luminiscence is directly proportional to the northern blot analysis.
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