RNA synthesized by MegaScript in vito transcription kit can be used for Rescue/over-expression study of gene?
I know that, most eukaryotic mRNA molecules have a 5' 7-methyl guanosine residue or cap structure which both functions in the protein synthesis initiation process and serves to protect the mRNA from intracellular nuclease digestion. Capped in vitro transcripts can be synthesized by substituting cap analog (m7G(5')ppp(5')G) for a portion of the GTP in the transcription reactions. In vitro transcripts which are to be microinjected into oocytes or other cells, or used for transfection experiments or in vitro splicing reactions, should be capped. However, for in vitro translation experiments it is frequently not necessary to cap in vitro transcripts. Now, I wonder, if I can use uncapped RNA for over expression study?
Hello Sharmin.
Not working. Follow the royal road.
There are original works that show less than >97% expression with uncapped RNA than capped RNA injection in Xenopus oocyte. So far many works also verified significantly low efficiency.
Oocytes from many species have no active roles in transcription process during early cleavage stage since they have maternally-derived mRNA to be translated by oocyte activation upon sperm penetration. Only at the zygotic genome activation, they may prepare necessary machinery for the transcription process of RNA synthesis to mature mRNA.
Uncapped RNAs are very unstable and degraded in the oocyte. You probably inject more RNA to increase efficiency, leading to intoxicating the oocytes.
Dear Dr. Fredrick and Dr. Lee thank you for valuable suggestions. Much appreciated indeed!
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