This observation suggests that the CDKN1A probe is sensitive to transcript level, but confirmation of this interpretation will require use of an alternative gene expression assay such as quantitative real-time PCR.
This provided us with empirical proof that our sorting strategy was sensitive to detecting transcripts dependent upon mtDNA status.
It is possible that subtractive hybridization may be more sensitive to detecting transcripts expressed at low levels.
Our results showed that primary cells are far more sensitive to SMILE transcript silencing than HeLa cells, as there was no need for Bortezomib treatment to induce XBP-1 overexpression in SMILE-silenced keratinocytes.
They are all based on the observation that lower k values make the assembler more sensitive to low-expressed transcripts, while larger k values make it more specific to high-expressed transcripts.
Importantly, computer profiling is especially sensitive to low-abundance transcripts that are normally underrepresented in cellular mRNA, such as transcription factors.
RNA-seq has three important advantages compared to microarray hybridization: it detects all existing transcripts irrespective of gene annotation, it is a direct quantitative measure of gene expression and it is more sensitive to lowly-expressed transcripts [ 34].
However, our earlier study did not establish whether Pea3 is upregulated prior to the formation of frank tumors, since Northern blotting was insufficiently sensitive to detect Pea3 transcripts in mammary glands prior to tumor development from either wildtype or MMTV/Wnt1 mice.
This indicated that amplified RNA was more sensitive to low abundance transcripts than the standard method using total RNA.
Of note, one round of amplification is demonstrated to be more sensitive to low abundance transcripts than using total RNA [ 18- 20].
