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[Preprint]. 2023 Oct 10:2023.10.10.561763.
doi: 10.1101/2023.10.10.561763.

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  1
Affiliations

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

Katherine M McKenney et al. bioRxiv. .

Update in

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 on 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 second, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the significant, reproducible reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of endogenous poly(A) binding protein, PABPC1, with poly-adenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, cost-effective method to enable researchers to detect and measure changes in RNA expression, processing, binding, and decay of RNAs.

Keywords: Biotinylation; Chemiluminescence; Northern blot; RNA; Streptavidin.

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Conflict of interest statement

Conflicts of Interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.. Overview of RNA detection by Chemi-Northern method.
The Chemi-Northern protocol consists of 5 steps: 1) Generation of biotinylated (purple pentagon) antisense nucleic acid probe (black line). As an example, an ethidium bromide stained denaturing formaldehyde-MOPS agarose gel of the firefly luciferase (Fluc) and nano-luciferase (Nluc) probes used in this study are shown on the right. An RNA size marker is included on the left, with sizes indicated in nucleotides (nt). 2) Extraction and purification of RNA (red line). As shown on the right, RNAs can be purified from a diverse sources using a variety of methods including organic phase separation (e.g. trizol reagent), spin column, or bead based methods (e.g. RNA coimmunoprecipitation assays). 3) Separation of RNA by charge to length ratio by denaturing formaldehyde-MOPS agarose gel electrophoresis. 4) Blotting transfer and immobilization of RNA to positively charged nylon membrane. 5) Visualization of target RNA hybridized to the biotinylated probe using streptavidin-HRP conjugate and enhanced chemiluminescence (ECL) detection. Gel, column, and test tube icons were created by Biorender.com under academic license agreement NE25MGJNLD.
Figure 2.
Figure 2.. Dynamic response range of mRNA detection by Chemi-Northern using biotinylated RNA probes.
HCT116 cells were transfected with increasing amounts (250, 500, 1000, and 2000 ng) of nano-luciferase reporter plasmid (Nluc), as indicated at the top. The ‘0’ ng condition indicates mock transfected cells with Nluc reporter. A total of 5 μg of purified cellular RNA was loaded into each lane of the denaturing formaldehyde-MOPS agarose gel and detected via Chemi-Northern using 50 ng/mL (A) or 500 ng/mL (B) of biotinylated antisense Nluc RNA probe. Chemi-Northern blot of expressed Nluc mRNA (639 nt +pA tail) at 1 sec exposure time is shown in the upper panels and ethidium bromide (EtBr) stain of ribosomal RNA (18S, 1869 nt and 28S rRNA, 5070 nt), serving as a loading control and indication of RNA integrity, are shown in the lower panels. (C) Graph showing the linear relationship between transfected reporter (x-axis) and signal intensity (y-axis) of Chemi-Northern blot at two different probe concentrations (50 and 500 ng/ml). Reported are the linear regression equation, y=mx+b and coefficient of determination, R2. All blots were quantified using AzureSpot Pro and graphs were created using GraphPad Prism software. (D) HCT116 cells were transfected with 1.25 μg of Nluc reporter plasmid and the purified total RNA was titrated (50, 500, 1000, 2500, and 5000 ng) and expressed Nluc mRNA was detected at 1 sec exposure by Chemi-Northern blot (upper panel) and EtBr stain (lower panel). (E) Quantitation and linear fit between total RNA mass (ng, x-axis) and signal intensity (y-axis). RNA was analyzed from Drosophila DL1 cells (F-G) or D.mel-2 cells (H-I) transfected with Nluc (806 nt +pA tail) or Fluc (1773 nt + pA tail) reporters using Chemi-Northern. Purified total cellular RNA was titrated as indicated at the top of the gels. Note that in panel F, the bottom panel shows the Drosophila rRNA species stained with EtBr, of which the 28S rRNA (3945 nt) is naturally processed into two fragments (1787 nt and 2112 nt) whereas the 18S rRNA is 1995 nt, as previously documented (30,31). (G, I) Quantitation of signal intensity relative to the amount of total cellular RNA analyzed.
Figure 3.
Figure 3.. Chemi-Northern using biotinylated DNA probes detects endogenous messenger and non-coding RNAs.
(A) Total RNA was purified from HCT116 cells and titrated (50, 100, 1000, 2500, and 5000 ng) onto a formaldehyde agarose gel followed by Chemi-Northern blotting using 5’ biotinylated DNA oligonucleotide antisense probes for endogenous β-actin (ACTB) mRNA at 85 sec exposure (1812nt +pA tail, upper panel) and 7SL ncRNA at 1 sec exposure (299 nt, middle panel). EtBr staining of ribosomal RNAs is shown in the lower panel. Quantitation and graphs showing linear fit of the total RNA mass (ng) versus signal intensity of the ACTB (B) and 7SL (C). (D) Total RNA of Drosophila DL1 cells titrated and endogenous 7SL (299 nt) and detected via Chemiluminescent Northern blot at 1 sec exposure. E) Quantitation of signal intensity relative to total cellular RNA.
Figure 4.
Figure 4.. Attomole sensitivity of Chemi-Northern detection.
(A) Ethidium bromide stained (EtBr) denaturing formaldehyde-MOPS agarose gel of 500 ng in vitro transcribed, purified synthetic Nluc mRNA and biotinylated Nluc antisense probe RNA (646 nt), confirmed proper size, purity, and quality. RNA size marker is included with nucleotide (nt) lengths indicated on the left. (B) Chemi-Northern detection of titrated synthetic Nluc mRNA at 6 sec exposure. The amount of the Nluc mRNA is indicated, with mass at the top and moles at the bottom. (C) Measured signal intensities in panel B are graphed relative to the amount of mRNA. Linear regression analysis was performed to assess the dynamic response, with the resulting line, equation, and coefficient of determination, R2.
Figure 5.
Figure 5.. Enhanced sensitivity by optimizing biotin incorporation into RNA probe.
(A) Ethidium bromided stained denaturing formaldehyde-MOPS agarose gel of Nluc 5ʹUTR RNA probes that were transcribed in vitro using the indicated ratios of biotinylated UTP (bioU) to unlabeled UTP. Total UTP concentration was the same in all reactions. (B) Chemi-Northern detection was performed on identical blots using Nluc 5ʹUTR probe with the indicated ratio of bioU:U. The gels were analyzed and imaged under identical conditions using the same exposure setting. 7SL RNA was detected using a biotinylated DNA oligo probe on the same blots, as a loading control. (C) Quantitation of the Chemi-Northern signals in panel B. Signal intensity for Nluc in each lane was normalized to its corresponding 7SL RNA signal. Fold change was then calculated relative to mean value for of the 1:2 bioU:U probe. Mean, SD, and the three data points for each condition are shown in the graph.
Figure 6.
Figure 6.. Chemi-Northern reproducibly measures mRNA regulation and binding by RNA-binding proteins.
(A) Regulation of the Nluc reporter mRNA with three wild type Pumilio Response Elements (3xPRE) in its 3′UTR was compared to the mutant version using Chemi-Northern. The PREs are specifically recognized by endogenous sequence specific RNA binding proteins, PUM1 and PUM2, which repress by causing degradation of the mRNA, as previously documented (21,24,25,32). Reporters were expressed in HCT116 cells. The Fluc mRNA served as a control for transfection efficiency. Nluc mRNA was detected at 5 sec exposure and Fluc mRNA at 10 sec. The 7SL RNA served as an internal control for equivalent loading of the gel and was imaged at 5 sec exposure time. (B) Quantitation of Chemi-Northern in panel A and three additional biological replicates. Log2 fold change of the Nluc 3xPRE reporter was determined relative to the mutant version, Nluc 3xPREmt, in each sample was calculated as described in the Materials and Methods. Mean and standard deviation values are plotted, along with the values of each of the replicates. Statistical significance of p = 0.0005, based on paired two-tailed t test, is indicated by ‘***’. (C) Nluc mRNA is enriched by RNA coimmunoprecipitation (RIP) of human RNA-binding protein PABPC1 from HCT116 cells. The top image shows the Chemi-Northern detection of the poly-adenylated Nluc mRNA at 40 sec exposure in the input samples and the robust enrichment in the PABPC1 RIP samples, but not the IgG negative control RIPs. The middle image shows the ethidium bromide (EtBr) stained denaturing formaldehyde-MOPS agarose gel from input and RIP samples. Ribosomal RNAs are clearly visible in the inputs, but not RIP samples. The image at the bottom shows the western blot of PABPC1 protein, demonstrating its presence in the input samples and enrichment in the RIP samples. Dashed vertical lines in the panels indicate that the images were cropped to show relevant lanes. For each image, all of the lanes are from the same blot and exposure. (D) Quantitation of the Chemi-Northern in panel C demonstrates significant, reproducible detection of Nluc mRNA in the PABPC1 RIP samples. Nluc RNA signal intensity in each RIP sample was normalized to its corresponding input sample, then the fold enrichment in the PABPC1 RIP was calculated relative to the IgG negative control. Mean and standard deviation values are plotted, along with the values of each of the three biological replicates. Statistical significance of p = 0.0005, based on paired two-tailed t test, is indicated by ‘***’.
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