Electrophoresis Of Rnas Using Formaldehyde Agarose Gels

1. Thoroughly clean an appropriate gel apparatus and combs by soaking them in 0.2% SDS solution overnight and washing them with a detergent. Completely wash out the detergent with tap water and rinse with distilled water three to five times.

2. Prepare a formaldehyde agarose gel mixture in a clean bottle or a beaker as follows:

Component Mini gel Medium gel Large gel dd.H2O 21.6 ml 54 ml 86.4 ml

Ultrapure agarose 0.3 g 0.75 g 1.2 g

Note: The percentage of agarose is normally 1% (w/v), but it can be adjusted with the sizes of the RNAs to be separated. The dd.H2O should be DEPC treated. Because the agarose is small in amount, its volume can be ignored in calculating the total volume.

3. Place the bottle (with the cap loose) or beaker in a microwave oven and slightly heat the agarose mixture for 1 min. Briefly shake the bottle to rinse any agarose powder stuck to the glass sides. To melt the agarose completely, carry out gentle boiling for 1 to 3 min, depending on the volume of agarose mixture. Gently mix, open the cap and allow the mixture to cool to 50 to 60°C at room temperature.

4. While the gel mixture is cooling, seal a clean gel tray at the two open ends with tape or a gasket and insert the comb in place.

5. Add the following components to the cooled gel mixture:

Components Mini gel Medium gel Large gel

10X MOPS buffer 3ml 7.5 ml 12 ml

Ultrapure formaldehyde 5.4 ml 13.5 ml 21.6 ml

Ethidium bromide (EtBr, 3 ml 7.5 ml 12 ml

6. Gently mix after each addition and pour the mixture into the assembled gel tray placed in a fume hood. Allow the gel to harden for 30 min at room temperature.

Caution: Formaldehyde is toxic; both DEPC and EtBr are carcinogenic. These reagents should be handled with care. Gloves should be worn when working with these chemicals. Gel running buffer containing EtBr should be collected in a special container designated for toxic waste disposal. Formaldehyde serves to denature any secondary structures of RNAs. EtBr is used to stain RNA molecules by interlacing in the regions of secondary structure of RNAs and fluoresces orange when illuminated with UV light. An alternative way is to stain the gel after electrophoresis is completed. The gel can be stained with EtBr solution for 10 to 30 min followed by washing in distilled water for 3 to 5 min. In addition, an appropriate volume of EtBr stock solution can be directly added to the RNA sample (1 ml of 1 mg/ml per 10 ml RNA) prior to loading.

7. While the gel is hardening, prepare the RNA sample in a sterile tube as follows:

Total RNA (10 to 35 mg/lane)

10X MOPS buffer, 3.5 ml

Ultrapure formaldehyde

Ultrapure formamide

Add DEPC-treated dd.H2O to 35 ml

8. Heat the tubes at 55°C for 15 min and immediately chill on ice to denature the RNAs. Briefly spin down afterwards.

9. Add 3.5 ml of 5X DEPC-treated loading buffer to the RNA sample. Proceed to sample loading.

10. Slowly and vertically pull the comb out of the gel and remove the sealing tape or gasket from the gel tray. Place the gel tray in an electrophoresis apparatus and add a sufficient volume of 1X MOPS buffer to the apparatus until the gel is covered to a depth of 1.5 to 2 mm above the gel.

Note: The comb should be slowly and vertically pulled out from the gel because any cracks occurring inside the wells of the gel will cause sample leaking when the sample is loaded. The top side of the gel where wells are located must be placed at the negative pole in the apparatus because the negatively charged RNA molecules will migrate toward the positive pole. If any of the wells contain visible bubbles, they should be flushed out of the wells using a small pipette tip to flush the buffer up and down the well several times. Bubbles may adversely influence the loading of the samples and the electrophoresis. The concentration of TBE buffer should never be lower than 0.4X. Otherwise, the gel may melt during electrophoresis. Pre-running the gel at a constant voltage for 10 min is optional.

11. Carefully load the samples, one by one, into appropriate wells in the submerged gel.

Notes: (1) A pipette tip should not be inserted all the way to the bottom of a well because this will likely break the well and cause sample leaking and contamination. The tip containing the RNA sample should be vertically positioned at the surface of the well, stabilized by a finger of the other hand and slowly loaded into the well. (2) The loading buffer should be at least 1X to final concentration; otherwise, the sample may float out of the well.

12. After all the samples are loaded, estimate the length of the gel between the two electrodes and set up the power supply at 5 to 15 V/cm of gel. Allow electrophoresis to proceed for 2 h or until the first blue dye reaches a distance of 1 cm from the end of the gel. If overnight running of the gel is desired, the total voltage should be determined empirically.

13. Stop electrophoresis and visualize the RNA bands in the gel under UV light. Photograph the gel with a Polaroid camera.

Caution: Wear safety glasses and gloves for protection against UV light. Notes: The purpose of taking a picture is to record different RNA molecules at different positions following electrophoresis, which is useful once a hybridized signal is detected. Pictures should be taken with a fluorescence ruler placed beside the gel. An appropriate exposure is needed to obtain two relatively sharp rRNA bands and a black background. For successful RNA separation on a gel, smear mRNA and sharp rRNA bands should be visible in each lane. The two sharp rRNA bands are 28S and 18S for animal RNAs or 25S and 18S for plant RNAs.

Materials Needed Microcentrifuge

Sterile microcentrifuge tubes (0.5 or 1.5 ml) Pipette or pipetman (0 to 200 ml, 0 to 1000 ml) Sterile pipette tips (0 to 200 ml, 0 to 1000 ml) Gel casting tray Gel combs

Electrophoresis apparatus DC power supply

Small or medium size of RNA electrophoresis apparatus, depending on samples

Ultrapure agarose powder

Total RNA samples

Specific DNA or RNA as a probe

Nylon membranes

3MM Whatman filters

Paper towels for blotting

UV transilluminator

Hybridization oven or shaker

Water bath with temperature control

Diethylpyrocarbonate (DEPC)

Formaldehyde (37%)


DEPC Water

0.1% (v/v) DEPC in dd.H2O, placed at room temperature overnight with a stir bar to inactivate any potential RNases. Autoclave to remove the DEPC.

10X MOPS Buffer

0.2 M 3-(N-morpholino)propanesulfonic acid (MOPS) 80 mM Sodium acetate 10 mM EDTA (pH 8.0)

Dissolve well after each addition in DEPC-treated dd.H2O. Adjust the pH to 7.0 with 2 N NaOH. Filter-sterilize and store at room temperature.

Ethidium Bromide (EtBr)

10 mg/ml in DEPC-treated dd.H2O, dissolve well and keep in a dark or brown bottle at 4°C.

Caution: EtBr is extremely toxic and should be handled carefully.

5X Loading Buffer 50% Glycerol 2 mM EDTA 0.25% Bromphenol blue 0.25% Xylene cyanol

Dissolve well in DEPC-treated water, filter-sterilize, and store at 4°C.

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