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…

開示

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

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記事を引用
Northern Blot to Detect and Quantify Specific MicroRNA in Total RNA Extract of Plant Tissue. J. Vis. Exp. (Pending Publication), e21371, doi: (2023).

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