IMR Press / FBL / Volume 2 / Issue 3 / DOI: 10.2741/A164

Frontiers in Bioscience-Landmark (FBL) is published by IMR Press from Volume 26 Issue 5 (2021). Previous articles were published by another publisher on a subscription basis, and they are hosted by IMR Press on imrpress.com as a courtesy and upon agreement with Frontiers in Bioscience.

Open Access Article
Experimental procedure for the detection of a rare human mRNA with the DIG System
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1 Boehringer Mannheim Customer Service Lab, International Support, Mannheim, Germany
Front. Biosci. (Landmark Ed) 1997, 2(3), 1–5; https://doi.org/10.2741/A164
Published: 15 February 1997
Abstract

Newcomers to the DIG System often inquire about the possibility of performing Northern blot hybridizations with nonradioactive techniques. With the following examples, we would like to share our protocol for performing highly sensitive Northern blots. This procedure strictly adheres to the standard procedures detailed in our manuals and pack inserts, and there are no special "tricks" required. As a target, we have used total human skeletal muscle RNA (Clontech). We selected two probes: beta-actin and a probe comprising the cDNA of the transcription factor CTF1, which expresses a low abundant mRNA. We used in vitro transcribed RNAs exclusively as probes because, during the development of the DIG System, we have found that RNA probes exhibit a 10-100-fold higher sensitivity with RNA targets than do DNA probes. They are also less prone to background problems caused by probe concentrations that are too high. For DNA probes, we recommend an optimal probe concentration of 25 ng/ml. Using a probe concentration that is even slightly too high (e.g., 1.5 fold) will dramatically increase the background. For RNA probes, we recommend an optimal probe concentration of 100 ng/ml, which will not lead to background problems. In the following examples, we describe all experimental details, starting from the gel run for the blot.

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