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

  1. Stop the pre-run and wash the wells before sample loading.
  2. 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.
  3. Complete loading of all the samples. Include loading the RNA decade marker.
  4. Run the gel at 80 V until the bromophenol blue dye runs almost completely. Bromophenol blue runs at 10 bp in a 15% denaturing acrylamide gel.

2. Preparation for electro-blotting

  1. Cut the N+nylon membrane to the dimensions of a glass plate and label the membrane at its top-right corner with an HB pencil.
  2. Gently place the membrane on the surface of sterile water, facing the labeled side towards the water surface. Pre-soak the membrane for 15 min.
  3. Cut 4 pieces of blotting paper I into the dimensions of a fiber pad.
  4. Prepare the gel sandwich by placing the gray side of the cassette down in a clean tray.
  5. Pre-wet the fiber pad and place it over the cassette. Remove air bubbles.
  6. Pre-wet one piece of blotting paper in 1x TBE and place it over the fiber pad. Remove air bubbles by rolling a plastic pipette over the paper. Lay another piece of pre-wet blotting paper and remove air bubbles.
  7. Carefully remove the gel from the running cassette and place it over the sandwich setup, such that the first loaded RNA sample is towards the right.
  8. Gently dip the pre-soaked membrane in 1x TBE and place it over the gel, facing the labeled side down. Roll out to remove the air bubbles.
  9. Dip a piece of blotting paper, lay it over the membrane, and remove the air bubbles. Dip another piece of blotting paper, place it over the sandwich setup, and remove air bubbles.
  10. Complete the sandwich by placing a pre-wet fiber pad over the setup and firmly closing the cassette.
  11. Place the transblot cassette in the module and fill 1x TBE, pH 8.2, up to the blotting mark.
  12. Transfer at 10 V, overnight at 4 °C.
  13. After transfer, place the damp membrane on a paper sheet and immediately cross-link the RNA to the membrane by irradiation with 254 nm UV light (120,000 µjoules/cm²). The cross-linked blot may be stored at 4 °C for further hybridization.

3. Preparation of radiolabeled probe

  1. Design a probe that is completely complementary to the small RNA which is to be detected.
  2. End label the probe using ƳP32ATP (obtained from BRIT) at its 5' end by combining the components as per the recipe provided in Table 1.
  3. Incubate the above reaction mixture at 37 °C for 30 min.
  4. Separate un-labeled ƳP32ATP from the probe by using a Sephadex G-25 column according to the manufacturer's protocol.

4.Hybridization of the blot

  1. Place the crossed-linked blot, RNA side facing the top, inside a hybridization bottle.
  2. Vigorously mix the ultra-sensitive hybridization buffer, add 10 mL of it, and incubate the blot inside a hybridization oven maintained at 35 °C, with rotation.
  3. Perform pre-hybridization for 20 min.
  4. Remove the bottle from the oven and gently add the labeled probe into the hybridization buffer.
  5. Hybridize the blot at 35 °C, with rotation for 12 h.
  6. After hybridization, carefully transfer the hybridization solution into 15 mL tube. This solution can be stored at 4 °C until re-use.
  7. Perform a quick wash of the blot for 2 min to remove excess hybridization solution by adding 2x SSC buffer plus 0.5% SDS. Discard the solution.
  8. Incubate the blot with 2x SSC buffer plus 0.5% SDS for 35 °C, with rotation for 30 min.
  9. Wash the blot again with 0.5x SSC buffer plus 0.5% SDS for 35°C, with rotation for 30 min.
  10. Place the blot inside a hybridization cover, remove the excess buffer, and seal it.
  11. Expose it to a radiation-free-phosphor imager screen overnight inside a cassette.
  12. Detect the hybridization signal using a biomolecular imager and analyze the results using suitable software.

Table 1: Table of Recipes

Buffer and solution Recipe Comments
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
Labeling of probe PNK buffer (10X, 2 µL) This mixture contains radioactive molecules, this step must be performed by trained personnel 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

Divulgaciones

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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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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