In recent years, it has been aptly used to validate and study the size and relative abundance of small noncoding RNAs. However, it has the unique advantage of providing information about the expression level and size of the transcript so that it can be used to detect RNA degradation and alternative splice product of the same gene or repetitive sequence motifs, to reveal deletions or errors in transcript processing, and to isolate novel transcripts from heterogeneous mRNA pools. Therefore, Northern blotting is not a desirable method for RNA analysis. Furthermore, Northern blotting is a relatively labor-intensive technique because the procedure consists of several steps. Ĭompared to qPCR, the major limitations of Northern blot analysis are low detection sensitivity and easy RNA degradation by contaminated exogenous ribonucleases (RNases) in the course of extensive handling of RNA prior to blotting. However, its prominent advantage of higher detection sensitivity also condemns the risk of the publication of inconsistent, irrelevant, and even misleading data based mostly on flawed qPCR results and associated interpretation because of the difference in the quality of RNA samples and the efficiency of reverse transcription (RT), and the inappropriate methodologies selected for the normalization and quantification of data. Among these three methods, qPCR is an often used technology in current molecular biology laboratories due to its extreme detection sensitivity, high specificity and throughput. Northern blot analysis, ribonuclease protection assay, and real-time polymerase chain reaction (qPCR) are the three most commonly used techniques for studying the expression of one or a small number of genes. The multitiered technological methods used for identification of gene expression patterns can be divided into three categories: techniques for detecting the expression of one or a small number of genes, whole transcriptome-based approaches, and targeted next generation sequencing assays for the selected specific sets of genes or genomic regions. The study of gene expression can provide us with knowledge about gene function and regulation. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. ![]() In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. ![]() If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
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