Dear Worm Breeders,
We have recently discovered that RNAi can produce drastically different
results depending on the template used to synthesize dsRNA. We
synthesized dsRNA from a 750 bp PCR fragment that corresponds to 2 exons
and a 53 bp intron (the primers are on exon/intron boundaries). This dsRNA
produced no phenotype (we sequenced it). However, RNAi using a full-length
cDNA produces early embryonic defects and lethality. We do not have
corresponding results for other genes, but we have injected dsRNA
corresponding to a wide variety of genes, many of which we expected to be
essential, and often (85%) do not see phenotypes.
We intend to compare cDNA templates to PCR genomic templates for a variety
of genes, but I would first like to survey all newsgroup readers for any
similar experiences. Perhaps the summation of all our experiences will
provide a tentative answer that can be confirmed quickly.
Do you have similar results using cDNA vs. genomic DNA as template for RNA
synthesis?
What percentage of genes inhibited by RNAi produce a phenotype?
Do you use cDNA as your RNA template or PCR of genomic fragments?
What are the sizes of the dsRNA and is there a correlation between whether
you see a phenotype?
If you use genomic fragments how many exons are included? Are they 5' or
3' exons?
I welcome suggestions for other variable to consider.
Please email responses to me at hunter at biosun.harvard.edu
Thank you,
Craig P. Hunter
Dept Molecular and Cellular Biology
Biological Laboratories
Harvard University
16 Divinity Avenue
Cambridge MA 02138
hunter at biosun.harvard.edu
office 617-495-8309
lab 617-496-0805
