Rhythmic transcripts from (B ) are plotted according to how the ratio of the mRNA amplitude divided by the pre-mRNA amplitude is changed between Dicer knockout and control (abscissa; the further to the left a transcript is located, the lower the amplitude becomes in Dicer knockouts) and according to the change in mRNA/pre-mRNA ratio on the ordinate, as in (C ).
No doubt, this bias leaves a large pool of undiscovered transcript variants with intronic polyadenylation sites.
This leaves a possibility of a fusion transcript as the result of our patient's deletion to be clarified by further experiments such as possibly 3' Rapid Amplification of cDNA Ends (RACE).
Summing up the number of conventionally detected transcripts and transcripts detectable by SR leaves a very small fraction of truly silent gene candidates.
Finally, any transcript for which the total number of reads was less than 44, i.e. an average of less than one transcript per lane, was culled from the dataset to leave a list of 46,446 transcripts.
The transcripts leave a great deal to be desired.
The RiboMinus depletion method eliminates fewer RNAs than polyA selection, leaving a larger number of transcripts in the overall pool.
In five cases, an abbreviated stop codon was inferred where cleavage of a downstream tRNA from the transcript would leave a partial codon of T or TA, such that subsequent mRNA polyadenylation could generate a TAA stop codon.
In five cases, an abbreviated stop codon was inferred where cleavage of a downstream tRNA from the transcript would leave a partial codon of T or TA, such that subsequent mRNA polyadenylation could generate a TAA stop codon; however, in each of these cases, if the reading frame extended through the first legitimate stop codon there would be only a short overlap with the downstream gene.
This is in accord with the viability of a deletion mutant for the pyrophosphohydrolase RppH [9], proposed to decap transcripts to leave a 5′ monophosphate terminus that would activate RNase E cleavage.
