Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Mar 18;30(4):448-462.
doi: 10.1261/rna.079880.123.

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules

Katherine M McKenney  1 , Robert P Connacher  1 , Elise B Dunshee  1 , Aaron C Goldstrohm  2
Affiliations

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules

Katherine M McKenney et al. RNA. .

Abstract

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated by denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 sec, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the reproducible, statistically significant reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of the poly(A) binding protein, PABPC1, with polyadenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, and cost-effective method to enable researchers to analyze expression, processing, binding, and decay of RNAs.

Keywords: RNA; biotinylation; chemiluminescence; northern blot; streptavidin.

PubMed Disclaimer

Update of

Similar articles

See all similar articles

References

    1. Alwine JC, Kemp DJ, Stark GR. 1977. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci 74: 5350–5354. 10.1073/pnas.74.12.5350 - DOI - PMC - PubMed
    1. Arvola RM, Chang CT, Buytendorp JP, Levdansky Y, Valkov E, Freddolino PL, Goldstrohm AC. 2020. Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs. Nucleic Acids Res 48: 1843–1871. 10.1093/nar/gkz1187 - DOI - PMC - PubMed
    1. Bohn JA, Van Etten JL, Schagat TL, Bowman BM, McEachin RC, Freddolino PL, Goldstrohm AC. 2018. Identification of diverse target RNAs that are functionally regulated by human Pumilio proteins. Nucleic Acids Res 46: 362–386. 10.1093/nar/gkx1120 - DOI - PMC - PubMed
    1. Bustin SA. 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29: 23–39. 10.1677/jme.0.0290023 - DOI - PubMed
    1. Chomczynski P, Mackey K. 1994. One-hour downward capillary blotting of RNA at neutral pH. Anal Biochem 221: 303–305. 10.1006/abio.1994.1416 - DOI - PubMed

LinkOut - more resources