Question

If you wanted to measure the levels of dpy-11 and bli-1 mRNA and protein in response to the RNAi treatment, how would you do it?

Answer 1

First of all isolate all RNAs from cell using specific method like Isolation of Rna by Trizole or other method. Check whether in your RNAs you have mRNAs by running sample on agarose gel and confirm the presence of mRNAs. Then you can use Real time PCR to detect the presence of dpy-11 and bli-1 mRNA. First amplify dpy-11 and bli-1 mRNA using specific primers for this m-RNA and then analyse your amplification if dpy-11 and bli-1 mRNA were present does not interfere by RNAi than their will be amplification of this m-RNAs takes place. This can be detect by flurophore which produce more fluroscence on amplification. If RNAi is interefering your m-RNA then their will be no amplification of m-RNA.

For detection of protein Use Western blotting technique (Outlines):-

First harvest cells then sonicate cells in lamilee buffer after that run SDS page separate all proteins of harvested cell according to thier molecular weight. Transfer this protein in Gel onto specific membrane like PVDF or Nitrocellulose.

After that Use antibody against collagen protein or dpy-11/bli-1 mRNA encoded protein than incubate overnight than after few washing of blot develop your blot using X-RAY film or chemiDocs.

In absence of RNAi you will get your protein band on your blot.