Additional File 5.

Aberrant TSS transcripts at Pol I, Pol II and Pol II genes. (a) qRT-PCR to quantitate transcripts initiated from a TSS upstream of the canonical TSS for IRF1, GAPDH and RN7SK (a Pol III gene) in shRNA-MEN1 and shRNA-NS cell lines. IRF1, GAPDH and RN7SK mRNA transcripts and aberrant TSS transcripts were normalized to β-actin; the aberrant TSS transcripts were calculated as a fraction of total mRNA, and presented as fold enrichment relative to the non-silencing shRNAmir condition. Error bars are standard error (n = 2). A student's one-tailed t-test determined significance. *P ≤ 0.05. (b) Effective qPCR primers could not be designed at the TSS of the 45S rRNA that is processed to 18S rRNA (RN18S1, a Pol I gene) and so endpoint PCR was used to attempt to detect aberrant TSS transcripts. 25 cycles of PCR did not generate an amplicon, nor did a second round of 25 cycles using an aliquot (10%) of the first reaction as template (arrow), but when genomic DNA was used a template an amplicon of the correct size was produced (positive control). This experiment was done for two biological replicate cDNA preparations, though only one is shown. 18S ORF primers detect the RN18S1 rRNA. RT+ indicates reverse transcriptase was included in the cDNA reaction while RT- indicates it was omitted.

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Auriemma et al. Epigenetics & Chromatin 2012 5:2   doi:10.1186/1756-8935-5-2