Analysis of Queuosine tRNA Modification Using APB Northern Blot Assay

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.

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

Bioscience Reports ◽

10.1007/bf01115042 ◽

1988 ◽

Vol 8 (3) ◽

pp. 255-261 ◽

Cited By ~ 13

Author(s):

Hans-Jürg Monstein ◽

Thomas Geijer

Keyword(s):

Caudate Nucleus ◽

Northern Blot ◽

Northern Blot Analysis ◽

Mrna Sequence ◽

Post Mortem ◽

Related Sequence ◽

Antisense Probe ◽

Highly Sensitive ◽

Northern Blot Assay ◽

Proenkephalin A

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.

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A versatile tRNA modification-sensitive northern blot method with enhanced performance

RNA ◽

10.1261/rna.078929.121 ◽

2021 ◽

pp. rna.078929.121

Author(s):

Abdul Khalique ◽

Sandy Mattijssen ◽

Richard J. Maraia

Keyword(s):

Northern Blot ◽

Assay Method ◽

Trna Modification ◽

Altered Gene Expression ◽

Trna Modifications ◽

Molecular Studies ◽

Quantitative Validation ◽

Base Modifications ◽

Altered Gene ◽

Positive Hybridization

The ~22 mitochondrial and ~45 cytosolic tRNAs contain several dozen different posttranscriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression and their deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our lab developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ~10 years ago which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of an DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32 and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.

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Detection of cytosolic tRNA in mammal by Northern blot analysis

Bangladesh Journal of Pharmacology ◽

10.3329/bjp.v12i3.32501 ◽

2017 ◽

Vol 12 (3) ◽

pp. 243 ◽

Cited By ~ 1

Author(s):

Alshammari Fanar Hamad ◽

Hye-Young Jeong ◽

Jong-Hun Han ◽

Irfan Ahmad Rather

Keyword(s):

Northern Blot ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Video Clip ◽

Solid Supports ◽

Physiological Conditions ◽

Transcriptional Alterations ◽

Trna Species ◽

Full Screen ◽

Northern Blot Assay

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.Video Clip of Methodology: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>

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RNA expression as a prognostic tool in idiopathic nephrotic syndrome

Orvosi Hetilap ◽

10.1556/oh.2007.27978 ◽

2007 ◽

Vol 148 (23) ◽

pp. 1067-1075

Author(s):

Krisztina Fischer ◽

Orsolya Galamb ◽

Béla Molnár ◽

Zsolt Tulassay ◽

András Szabó

Keyword(s):

Nephrotic Syndrome ◽

Northern Blot ◽

Idiopathic Nephrotic Syndrome ◽

Minimal Change ◽

Ribonuclease Protection Assay ◽

Rna Expression ◽

Rt Pcr ◽

Protection Assay ◽

Ribonuclease Protection

A gyermekkori nephrosis 90%-a idiopathiás nephrosis szindróma. Az idetartozó három kórkép, a minimal change betegség, a mesangialis proliferatio és a focalis sclerosis hasonló klinikai képpel jelentkező, eltérő prognózisú és terápiás válaszú betegség. Dolgozatunk célja az idiopathiás nephrosis szindrómába tartozó kórképek kialakulásával, progressziójával összefüggő genetikai ismeretek, génexpressziós változások áttekintése és funkcionális csoportosítása. A génexpressziós változások meghatározásának eszközeként, dolgozatunk röviden összefoglalja a northern blot, a ribonuclease protection assay, az in situ RNS-hibridizáció, a kvantitatív RT-PCR és a microarray módszerek lényegét. Az eddig elvégzett vizsgálatok a DNS-szintézis és repair gének, növekedési faktorok, extracelluláris mátrix, extracelluláris ligandreceptorok, extracelluláris jelátvitel zavarai mellett kiemelik a metabolikus és transzporter gének, illetve az immunszabályozó gének molekuláris eltéréseit, amelyek összefüggésben vannak az idiopathiás nephrosis szindróma eddig megismert molekuláris hátterével. A chiptechnológia fejlődésével és elterjedésével ezek a markerek és a hagyományos vizsgálati módszerek párhuzamos alkalmazása rutindiagnosztikai szempontból is fontossá válhat.

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