RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

Hi there. So this is a small RNA northern protocol. It’s a derivative of protocol originally optimized by Dr.Rashid Akbergenov.

In this method, one can detect small RNA’s of anywhere between 20-24 nucleotide in length, very precisely and at very high resolution. The protocol is basically a modified form of traditional Northern analysis, but here, the percentage of acrylamide is quite high, it’s 15 percent. This northern can be used for detecting already known small RNA, either micro RNA or other set of small RNA, when the sequence is known or it can be used for confirmation of abundance of small RNA coming from next generation sequencing datasets.

Advantage of this method is that, one can not just look at micro RNA abundance, but also the abundance of precursors, for example micro RNAs may leave out a precursor which is around 30 to may be a few hundred nucleotide long, this can be also detected simultaneously, along with the micro RNA. The protocol is very simple, very straight forward, but one has to be very cautious while performing some critical steps which are listed in the end of this protocol. For preparation of 10 mL gel mix, add 4.8 grams of urea, 3.75 mL of 40%Acrylamide solution, and 1 mL of freshly prepared 10X TBE, pH 8.2 and gently mix the solution.

Dissolve urea completely, by heating the mixture in a water bath, until the solution looks clear. Make up the volume to 10 mL, using sterile MilliQ water and cool the gel mix to room temperature. Wash the required apparatus with detergent, scrub them gently to remove residual TBE and Acrylamide.

Further rinse them with MilliQ water and allow it to dry. Assemble the glass plate together, ensure both are at the same level. Place them firmly on the sponge.

Add 8 micro liter of TEMED and 80 micro liter of 10%freshly prepared APS to the gel mix. Gently mix and pour the gel mix to the assembled plates. Ensure that the gel does not leak.

Place the comb carefully. Allow the gel to polymerize for approximately 45 minutes. After polymerization of the gel, remove the glass plates from the assembly.

Wash the glass plates with MilliQ water. And place them in the running cassette. Place the running cassette inside the tank.

Pour freshly prepared 1X TBE, pH 8.2. Remove the comb carefully. Wash the wells of the gel thoroughly by pipetting the buffer.

This step removes precipitates of urea from the well and eases the RNA to run uniformly across the gel. Close the lid of the apparatus and pre-run the empty gel at 80 volts for 30 minutes, to check for any buffer leakage. For the preparation of 10 mL loading dye, weigh 5 mg of bromophenol blue, 5 mg of xylene cyanol and add 10 mL of deioinised formamide and mix them well.

Aliquot the dye and store at 4 degree for further use. Aliquot 10 microgram of total RNA into sterile 1.5 mL tubes. Make sure that the quality of the RNA, i.e.

the 260:230 ratio is greater than or closer to two. Place the tube inside a speedVac and vacuum dry the samples. Re-suspend the dried RNA samples in 8 micro liter of loading dye.

Heat the samples at 98 degrees for two minutes. Cool them for one minute at room temperature. Vortex the cooled RNA samples to ensure proper re-suspension in the loading dye.

Spin the tubes. Repeat the steps of heating, cooling and vortex for three times for getting free flowing RNA. Stop the pre-run of the gel.

Wash the wells thoroughly, before loading the sample, to remove deposits of the urea inside the wells. Heat the re-suspended RNA samples at 98 degrees for one minute. Load the samples hot into the wells using capillary tips.

Avoid introducing air bubbles. Complete loading of all the samples and assemble the lid. Run the gel at 80 volts for three hours or until the bromophenol blue runs completely.

Use positively charged nylon membrane for the transfer. Cut it to the dimensions of the glass plate. Label the membrane at it’s top right corner.

Gently place the membrane on the surface of sterile MilliQ water make sure to place the label side downwards, facing the water surface. Take a clean tray and prepare gel sandwich for electro transfer. Place the gray side of the cassette down.

Pour 1X TBE slightly above the level of the cassette. Pre-wet the fibre pad in 1X TBE and squeeze it to remove air bubbles. Cut two pieces of blotting paper to the size of fibre pad.

Pre-wet a piece of blotting paper in 1X TBE and place it over the fibre pad. Remove air bubbles by rolling a plastic pipette over the paper. Pre-wet another piece of blotting paper in 1X TBE and lay it over the cassette.

Roll over to remove air bubbles. Now the sandwich setup is ready for electro transfer. After completion of the electrophoresis, stop the run and remove the lid from the apparatus.

Remove the running cassette from the setup. Take out the glass plates from the running cassette. Carefully remove the gel from the assembly.

Lay it over the blotting paper such that the first loaded RNA sample is towards your right. Gently dip the pre-soaked membrane in 1X TBE and place it over the gel, facing the labeled side down. Do not allow the membrane and gel to dry.

Roll over gently to remove air bubble. Dip one piece of blotting paper in 1X TBE and lay it over the membrane. Remove air bubbles.

Place another piece of blotting paper and remove the air bubbles. Complete the sandwich by laying the fibre pad over the assembly. Close the cassette firmly.

Place the trans-blot cassette in the module. Fill the tank with 1X TBE, pH 8.2, up to the blotting mark. Close the lid of the apparatus and transfer at 10 volts overnight at 4 degrees or in a cold room.

Keep the UV cross linker ready and set it to 1200, just before completion of electro transfer. After completion of transfer, remove the cassette from module. Place the damp membrane on a A4 sheet, placing the marked side upwards.

Cross-link the RNA to the membrane by irradiation with 254 nanometre UV light at 120 milli joules. The cross-linked blot may be stored at four degree or used for hybridization. Design a probe that is completely complimentary to the small RNA that has to be detected.

End label the probe at it’s five prime end using PNK enzyme and combine the components as shown. Incubate the reaction mixture at 37 degrees for 30 minutes. After 30 minutes of incubation use G-25 columns to separate unlabeled probes from the reaction mixture.

For improved labeling of the probe, prepare the G-25 column before use. Place the blot, RNA side facing top, inside a hybridization bottle. Vigorously mix hybridization buffer before use.

Add 10 mL of hybridization buffer inside and place the hybridization bottle inside the hybridization oven, maintain at 35 degrees with rotation. Perform pre-hybridization for 20 to 30 minutes. After pre-hybridization remove the bottle from oven.

Add the labeled probe into the hybridization buffer gently. Make sure air bubbles are not created. Incubate the blot inside the oven at 35 degrees with rotation for 12 hours.

After hybridization, remove the hybridization buffer from the bottle. Perform a quick wash of the blots, using wash buffer-I. This step is performed to remove excess hybridization solution from the blots.

Further incubate the blots at 35 degrees for 30 minutes using wash buffer-I. Perform another wash using buffer-II at 35 degrees for 30 minutes. After washing of the blot, place it inside a hybridization cover, remove excess buffer and seal it.

Place it inside a cassette and expose it to a radiation free phospho-imager screen for 12 hours. Detect the hybridization signal using typhoon biomolecular imager and analyze the results using ImageJ software. For hybridization of the blot with other probes, perform stripping steps as illustrated.

With this technique one can estimate the abundance of micro RNA, as well as it’s length. From this image, expression of micro RNA 397 can be detected in all the samples. In this blot, the abundance of sample one is approximately 5 reads per million, as per next generation sequencing data, suggesting that our technique can detect less abundant micro RNAs too.

In addition, by using ImageJ quantification software, one can quantify the expression of micro 397 among the samples. Here we have used U6 and micro RNA 168 as loading controls. I will be talking about some crucial steps that must be considered while performing the experiment.

Make sure to use good quality RNA for sample preparation. While vacuum drying the samples, avoid over drying them. Re-suspension of RNA in the loading dye is critical.

Ensure to prepare a free flowing sample. Care must be taken while loading the gel. The wells of the gel must be washed and the sample have to be loaded in a straight line inside the wells.

During the electro transfer, spreading of the membrane in water, just before dipping it in 1X TBE is essential, it improves the transfer. The hybridization of the blot must be performed at 35 degrees. Maintain the temperature of the hybridization oven.

For repeated use of the blots, membranes must be stored at 4 degree. They should be kept damp in 2X SSE. Unlike other PCR based methods, this method ensures quantification of the expression, as well as the determination of the size of the micro RNAs.