Northern Blot to Detect and Quantify Specific MicroRNA in Total RNA Extract of Plant Tissue
Published: May 31, 2023
Abstract
Source: Tirumalai, V., et al. RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs. J. Vis. Exp. (2020)
In this video, we demonstrate the northern blot, a hybridization technique to detect and quantify microRNAs (miRNAs) from total RNA extracted from plant tissue.
Protocol
1. Gel electrophoresis Stop the pre-run and wash the wells before sample loading. Heat the samples at 98 °C for 1 min and load the samples hot into the well using capillary tips. Insert the tip into the bottom of the well so that the sample occupies one thin layer in the well. Complete loading of all the samples. Include loading the RNA decade marker. Run the gel at 80 V until the bromophenol blue dye runs almost completely. Bromophenol blue runs at 10 bp in a 15% denaturing…
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The authors have nothing to disclose.
Materials
| 40% Acrylamide-bisacrylamide solution | Sigma | A9926 | Chemical |
| Ammonium persulphate (APS) | BioRad | 1610700 | Chemical |
| Blotting paper | Whatman blotting paper I | 1001-125 | Assay |
| Bromophenol blue | Sigma | B5525-5G | Chemical |
| Capillary loading tips | BioRad | 2239915 | Plastic ware |
| Deionized formamide | Ambion | AM9342 | Chemical |
| Heating block | Eppendorf | 5350 | Device |
| Hybond N+nylon membrane | GE | RPN203B | Assay |
| Hybridization bottle | Sigma | Z374792-1EA | Plastic ware |
| Hybridization Oven | Thermo Scientific | 1211V79 | Device |
| N,N,N',N'- Tetramethylethylenediamine (TEMED) | Sigma | T7024-25ml | Chemical |
| PhosphorImager | GE- Typhoon scanner | 29187194 | Device |
| PhosphorImager screen and cassette | GE healthcare | GE28-9564-75 | Device |
| Pipettes | Gilson, models: P20 and P10 | FA10002M, FA10003M | Plastic ware |
| Plastic pipette | Falcon | 357550 | Plastic ware |
| Polyacrylamide gel apparatus | BioRad | 1658003EDU | Device |
| Sephadex G-25 column | GE healthcare | 27532501 | Device |
| Speed Vac Concentrator | Thermo Scientific | 20-548-130 | Device |
| Spinwin | Tarsons | 1010 | Device |
| T4 Polynucleotide Kinase (PNK) | NEB | M0201S | Assay |
| Transblot apparatus | BioRad | 1703946 | Device |
| ULTRAHyb hybridization buffer | Ambion | AM8670 | Assay |
| Urea | Fischer Scientific | 15985 | Chemical |
| UV-crosslinker | UVP | CL-1000L | Device |
| Vortex | Tarsons | 3020 | Device |
| Water bath | NEOLAB | D-8810 | Device |
| Xylene cyanol | Sigma | X4126-10G | Assay |
