Detection of cytosolic tRNA in mammal by Northern blot analysis

Abstract

<p>Out of entire cascade of technologies and strategies, Northern blot assay remains the most preferential approach for immediate and accurate evaluation of expressed RNA species. However, an abundance of tRNAs species under physiological conditions compared to other small RNAs makes it difficult to accurately evaluate their transcriptional alterations through traditional Northern blot assay. Here, we describe an efficient protocol for detecting subtle alterations in tRNA species in mammals by a modified Northern blot assay. This report also compares the chemical versus UV-based crosslinking of tRNA species to the surface of solid supports.</p><p><strong>Video Clip of Methodology</strong>:</p><p>Detection of cytosolic tRNA in mammal by Northern blot analysis: 16 min 55 sec <a href="https://youtube.com/v/6D2PdAprecE">Full Screen</a> <a href="https://youtube.com/watch?v=6D2PdAprecE">Alternate</a></p>

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“…Collect and combine the aqueous phase, the RNA was precipitated by adding 3 volume of ice-cold ethanol, and wash twice with ice-cold 75% ethanol. Northern blots were performed according to a previous report with minor modifications ( Hamad et al., 2017 ). Briefly, 1 ug tRNA enriched small RNA was loaded onto an 8 M urea-denatured PAGE gel and run in 1× TBE with a constant voltage of 80 V. The RNA was transferred to a positively charged nylon membrane and hybridized using a specific probe.…”