Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes

Nianhui Zhang,Mingdi Zhang,Tao Yang

doi:10.1186/s12864-021-08275-w

Abstract

BackgroundNorthern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use.ResultsIn this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. 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. 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. 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. 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. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. 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.ConclusionsThe modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA.

Highlights

Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods
Improved detection sensitivity by modification of posthybridization washes: quantitatively controlled moderate‐stringency washes The principle of detecting specific mRNA in heterogenous mRNA pools by Northern blot analysis is based on the ability of the complementary single-stranded nucleic acid probe to form hybrid molecules with the target, in which after removal of the unbound and nonspecific bound probes, the specific bound probes on the membrane are detected by an appropriate detection technique [4, 10]
Such prolonged washing can cause nonspecific and specific bound probes to be washdown from the membrane, which may be worse when detecting the expression of low expression genes. One of such examples is the detection of the expression of Aox1, a gene expressed in low-copy number that encodes the mitochondrial alternative oxidase (AOX), which is a regulatory component embedded in the inner face of the mitochondrial inner membrane [21]

Summary

Introduction

Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. 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 [1, 3, 4]. 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 [1, 3, 5, 6]. It is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use

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