Invitro Transcription - size distribution of transcripts - (May/28/2008 )
I would like to know that for a given gene of ~ 1Kb size that is subjected to invitro transcription:
a) what fraction of the m-RNA products are full length
what is the size distribution of m-RNA products obtained (truncated/longer/etc.,)
c) how does the size distribution of m-RNA products in invitro transcription change with gene size (1kb versus 4KB gene).
any input will be helpful.
What system are you using for in vitro transcription?
Rabbit reticulocyte lysate will give you a reasonable level of full length.
Hela or other nuclear extracts will give you almost 0% full length. Almost all will be under 500 bp.
That's why people insert a tag for primer extension or using long inserts of "G" so that radiolabed "G"s can be incorporated into the mRNA.