This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.
MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.
The recent discovery of small regulatory RNAs, microRNAs, has led research in understanding them and their role in plants and animals1. Long precursors of miRNAs are processed into 21 to 24 nt mature miRNAs by HYL1 and specific dicer-like proteins2,3. A 22 nt miRNA can initiate cascade silencing by generating secondary siRNAs4. Studies have shown the role of miRNAs and secondary siRNAs in development, cell fate and stress responses5,6.
Northern hybridization is an experimental method routinely employed to detect specific RNA molecules. This method customizes its use in the detection of approximately 19 -24 nt long small RNAs from a pool of total RNAs7. In this demonstration, we illustrate the use of this technique for the detection and quantification of miRNAs. This method uses labelling of probes using radioisotopes; thus, miRNA levels in the sample can be detected with increased sensitivity. Unlike PCR-based methods, this method ensures quantification of expression as well as size determination of the miRNAs. In this protocol, we show crucial steps that improve miRNA detection. We have modified steps in blotting and hybridization for obtaining high resolution signal detection of miRNAs. This technique can also be used for the detection other endogenous small RNAs such as siRNAs, tasi-secondary RNAs and snoRNAs.
1. Preparation of a 15% denaturing polyacrylamide gel
2. Assembly of the glass plates and electrophoresis unit
3. Preparation of loading dye and samples
4. Gel electrophoresis
5. Preparation for electro-blotting
6. Preparation of radiolabeled probe
7. Hybridization of the blot
In this demonstration, we have detected and quantified the expression of miR397 in different tissues of indica rice var whiteponni (Figure 1). miR397 is a 22 nt miRNA and conserved miRNA. The expression of miR397 can be detected in all the tested samples. As per the next-generation sequencing data, sample 1 (seedling tissue) has miR397 at 5 reads per million (rpm). We detected its signal comfortably, indicating that the method is very sensitive and can be used to detect even very low abundant miRNAs. In this experiment, we have used miR168 and U6 as loading controls.
In this blot (Figure 1), strength of signals was quantified using ImageJ. The expression of miR397 is highest in vegetative leaf sheath.
Figure 1: Detection and quantification of expression of miR397 in different tissues of rice samples. Please click here to view a larger version of this figure.
Buffer and solution | Recipe | Yorumlar |
10 X TBE | 0.89M Tris buffer | pH should be set to 8.2 using acetic acid |
0.89M Boric acid | ||
30mM EDTA | ||
Gel mix | 15% acrylamide-bisacrylamide sol, 19:1 | Gel mix should not contain urea crystals |
8M urea | ||
1X TBE | ||
Gel loading dye | 0.05 %(w/v) bromophenol blue | Care should be taken while handling deionized formamide |
0.05 %(w/v) Xylene xyanol | ||
100 % deionized- formamide | ||
Labelling of probe | PNK buffer (10X, 2 µL) | This mixture contains radioactive molecules, this step must be performed by trained personel inside a radioactive lab |
PNK enzyme (1 µL) | ||
Oligo (100 µM, 0.1 µL) | ||
ƳP32 ATP (4 µL) | ||
Wash Buffer – I | 2X SSC | |
0.5% (w/v) SDS | ||
Wash Buffer – II | 0.5X SSC | |
0.5% (w/v) SDS | ||
Stripping Buffer – I | 0.5X SSC | |
0.1% (w/v) SDS | ||
Stripping Buffer – II | 0.1X SSC | |
0.1% (w/v) SDS |
Table 1: Table of recipes.
This method can be extensively used for detection and quantification of small RNAs including less abundant miRNAs. The protocol mainly describes the steps for denaturing the total RNA in a loading buffer, size separation by gel electrophoresis, transfer of RNA to a membrane, cross-link the RNA onto membrane and hybridize using desired radiolabeled oligo probes.
The critical step for any blotting experiment is the use of good quality RNA for sample preparation. Before loading the gels, one must make sure that the samples are free flowing and not sticking to the loading tips. Care must be taken while loading the sample, tip should be inserted just above the bottom of the well so that sample occupies one thin layer in the well. The temperature of hybridization oven must be maintained at 35 °C for detection of miRNAs that are extremely less abundant. For repeated hybridization of blot, store the membrane at 4 °C by keeping it damp in 2x SSC.
This method can be used to detect small RNAs from tissues that are rich in polysaccharides and polyphenols8. In this protocol, usage of vacuum drying for concentrating RNA samples provides better stability and less loss of sample compared to other older methods9. Other modification in the method includes, spreading of the membrane in water before dipping in 1x TBE during electro-transfer. This improves the efficiency of RNA transfer, providing better resolution of blot.
A major limitation of the method is usage of radioisotopes, which needs trained personnel and a radioisotope lab to perform the hybridization experiments. This method here provides detailed information regarding all the steps involved in the RNA blot analysis for the detection of miRNAs. This protocol also ensures the size of the small RNA apart from its signal detection10. The technique provides a robust tool for molecular biologists to estimate the abundance of various small RNAs such as miRNAs, secondary siRNAs and snoRNAs.
The authors have nothing to disclose.
The authors acknowledge the access to radiation lab provided by the host institute and BRIT for radioisotope. PVS laboratory is supported by National Center for Biological Sciences, Tata Institute for Fundamental Research and grants (BT/PR12394/AGIII/103/891/2014; BT/IN/Swiss/47/JGK/2018-19; BT/PR25767/GET/119/151/2017) from Department of Biotechnology, Government of India. MP acknowledges DBT-Research Associateship, DBT, Government of India.
40% Acrylamide-bisacrylamide solution | Sigma | A9926 | |
Ammonium persulphate (APS) | BioRad | 1610700 | |
Blotting paper | whatmann blotting paper I | 1001-125 | |
Bromophenol blue | Sigma | B5525-5G | |
Capillary loading tips | BioRad | 2239915 | |
Deionized formamide | Ambion | AM9342 | |
Heating block | Eppendorff | 5350 | |
Hybond N+nylon membrane | GE | RPN203B | |
Hybridization bottle | Sigma | Z374792-1EA | |
Hybridization Oven | Thermo Scientific | 1211V79 | |
N,N,N’,N’-Tetramethylethylenediamine (TEMED) | Sigma | T7024-25ml | |
PhosphorImager | GE- Typhoon scanner | 29187194 | |
PhosphorImager screen and cassette | GE healthcare | GE28-9564-75 | |
Pipettes | Gilson, models: P20 and P10 | FA10002M, FA10003M | |
Plastic pipette | Falcon | 357550 | |
Polyacrylamide gel apparatus | BioRad | 1658003EDU | |
Sephadex G-25 column | GE healthcare | 27532501 | |
Speed Vac Concentrator | Thermo Scientific | 20-548-130 | |
Spinwin | Tarsons | 1010 | |
T4 Polynucleotide Kinase (PNK) | NEB | M0201S | |
Transblot apparatus | BioRad | 1703946 | |
ULTRAHyb hybridization buffer | Ambion | AM8670 | |
Urea | Fischer Scientific | 15985 | |
UV-crosslinker | UVP | CL-1000L | |
Vortex | Tarsons | 3020 | |
Water bath | NEOLAB | D-8810 | |
Xylene cyanol | Sigma | X4126-10G |