Zooming in: PAGE-Northern Blot Helps to Analyze Anti-Sense Transcripts Originating from Human rIGS under Transcriptional Stress

Abstract

Ribosomal intergenic spacer (rIGS), located between the 45S rRNA coding arrays in humans, is a deep, unexplored source of small and long non-coding RNA molecules transcribed in certain conditions to help a cell generate a stress response, pass through a differentiation state or fine tune the functioning of the nucleolus as a ribosome biogenesis center of the cell. Many of the non-coding transcripts originating from the rIGS are not characterized to date. Here, we confirm the transcriptional activity of the region laying a 2 kb upstream of the rRNA promoter, and demonstrate its altered expression under transcriptional stress, induced by a wide range of known transcription inhibitors. We managed to show an increased variability of anti-sense transcripts in alpha-amanitin treated cells by applying the low-molecular RNA fraction extracted from agarose gel to PAGE-northern. Also, the fractioning of RNA by size using agarose gel slices occurred, being applicable for determining the sizes of target transcripts via RT-PCR.

Keywords

• northern blot
• non-coding RNA
• small RNA
• rRNA
• rIGS
• rRNA depletion