Detection of cytosolic tRNA in mammal by Northern blot analysis

<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>

Intervening Sequence Acquired by Lateral Gene Transfer in Tropheryma whipplei Results in 23S rRNA Fragmentation

ABSTRACT Completion of Tropheryma whipplei genome sequencing may provide insights into the evolution of the molecular mechanisms underlying the pathogenicity of this microorganism. The first postgenomic application was the successful design of a comprehensive culture medium that allows axenic growth of this bacterium, which is particularly recalcitrant to cultivation. This achievement in turn permitted analysis of T. whipplei RNA without contaminating eukaryotic nucleic acids. To obtain high-quality RNA, several extraction methods were compared, but under all conditions tested an atypical profile was observed. By using a Northern blot assay we demonstrated that an insertion sequence previously described in T. whipplei 23S rRNA is in fact an intervening sequence excised during maturation. This cleavage could involve an RNase III identified in the genome of this microorganism. Among the bacteria with a 23S rRNA insertion sequence, T. whipplei is the only gram-positive microorganism. We present phylogenetic evidence that this mobile genetic element was acquired by lateral gene transfer from another enteric bacterium.

Elevated expression of heat-shock protein gene in the fibroblasts of patients with scleroderma

1. Scleroderma is a systemic autoimmune disorder characterized by fibrosis affecting the skin, lung, kidney and other organs. The 70 kDa heat-shock protein has been implicated as essential for cell function during cell growth and differentiation. To study the molecular basis of intracellular events in sclerotic fibroblasts, we compared the expression of the hsp 70 gene in sclerotic and normal control fibroblasts by a run on nuclear transcription assay and a Northern blot assay. 2. In the run on nuclear transcription assay, sclerotic fibroblasts expressed an approximately eightfold higher level of hsp 70 transcription than normal fibroblasts in the quiescent condition after serum starvation. After stimulation with serum, the transcription level of the hsp 70 gene was almost similar in sclerotic and normal control fibroblasts. 3. In the Northern blot assay, the hsp 70 gene transcript, which was present in increased amounts in sclerotic fibroblasts, exhibited normal size.

A highly sensitive northern blot assay detects multiple proenkephalin a-like mRNAs in human caudate nucleus and pheochromocytoma

Total RNA from post mortem human caudate nucleus, cerebellum, cerebral cortex and pheochromocytoma tissues has been prepared. Northern blot analysis, using a single-stranded human proenkephalin A antisense probe (cRNA), revealed the existence of two different proenkephalin A-like sequences in the human caudate nucleus and pheochromocytoma RNA extracts of approximately 1400 and 1000 nucleotides in length respectively, whereas no specific RNA bands could be detected in the cortex and only the 1400 nucleotide band was present in the cerebellum. Under highly stringent hybridization conditions, the proenkephalin A-like RNA bands still appear, indicating that the detected RNA species have either identical or a closely related sequence to that of the wellcharacterized human proenkephalin A mRNA sequence.