Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Ethical approval for this study was obtained from Shiraz University of Medical Sciences (Approval number: 93-01-01-71783-07-2014).

NOTE: Use male Sprague–Dawley rats weighing 250 g. Place the vial containing a sliver of pancreatic tissue immersed in RNA stabilizing reagent in a liquid nitrogen tank at -80 °C and use RNA extraction reagent solution to maintain the integrity of RNA.

1. Removal of the rat pancreatic tissue

1. Prepare the operating room and place all required materials under the hood.
2. Sterilize all the surgical instruments (Dressing forceps, Brown-Adson forceps, Iris scissors, and Littler scissors) in an oven for at least 4 h at 240 °C to inactivate RNases¹⁵. Sterilize the surface of the surgery place with 70% alcohol under the hood.
3. Inject ketamine/xylazine intraperitoneally using an insulin syringe [80/8 mg/kg]. Check the depth of anesthesia by pinching the rat’s toes for a lack of response.
4. Add 1 mL of RNA stabilization reagent in the proper microtube (2 mL) to inhibit activation of endonucleases in excised tissues.
5. Immediately place the rat on the surgical board (head away from the surgeon) for a mid-line incision.
6. Sterilize the entire abdominal surface with 70% alcohol and remove rat hair using a hair clipper with #40 blades.
7. Make a V-shape incision to open the abdomen from the pubic area to the front legs with Dressing forceps, Brown-Adson forceps, Iris scissors, and Littler scissors.
8. Flip the abdominal organs to the left side to expose the pancreas. Carefully find the pancreatic tissue, which spreads in the abdominal cavity. Locate the area under the spleen to find the pancreas without being confused with the fat tissue.
9. Immediately remove 20-30 mg of pancreas from the abdominal cavity in less than 2 min. Put the removed tissue in the 2 mL microtube quickly and cut it with a sterile cutter to penetrate the separated tissues with RNA stabilization reagent.
10. Place the microtube in a nitrogen tank to freeze immediately. Keep the microtube in a -80 °C freezer for 24 h¹⁶.
11. After 24 h, transfer the tiny pieces of pancreatic tissues to an ice container.
12. Inject potassium chloride (KCl) intracardially for euthanasia of rats after surgery.

2. RNA extraction

1. Sterilize the surface under the hood with 70% alcohol.
2. Put 1 mL of the RNA extraction reagent, which contains guanidinium thiocyanate to inhibit RNase, in the sterile microtube.
3. Transfer the separated pancreatic tissue to the microtube. Transfer the microtube to the liquid nitrogen to freeze immediately.
4. Homogenize the tissue with the micro tip probe sonicator at 4 °C set to level 20 for 60 s on and 5 s off.
5. Incubate each homogenized sample for 5 min at 4 °C using crushed ice to allow the nucleoprotein complexes to be completely dissociated.
6. Enrich the samples with 0.2 mL of chloroform for each 1 mL of RNA extraction reagent. Cap the tube firmly and shake it forcefully for 15 s.
7. Incubate the tube for 15 min on the crushed ice (4 °C) to separate the reagent in three phases.
8. Run the centrifuge at 12,000 x g for 15 min at +2 to +8 °C. Transfer the colorless aqueous phase to a new microtube.
9. Add 0.5 mL of 100% isopropanol to the aqueous phase. Close the tube and then invert it at least 3 times to mix the RNA thoroughly.
10. Incubate the sample for 5-10 min on a cold box (4 °C) to allow RNA precipitation.
11. Run the centrifuge at 12,000 x g for 15 min at +2 to +8 °C. Discard the supernatant.
12. Enrich each of the centrifuge tubes with 1 mL of 75% ethanol.
13. Wash the RNA pellet by inverting the tube at least 3 times.
14. Run the centrifuge at 7,500 x g at +2 to +8 °C for 5 min. Discard the supernatant to remove the excess ethanol from the RNA pellet by air-drying.
15. Re-suspend the RNA pellet in diethylpyrocarbonate (DEPC)-treated RNase-free water.
16. Pass the solution through a pipette tip several times to dissolve the RNA pellet. Then, incubate the solution for 10-15 min at +65 °C.

3. Evaluating RNA Integrity with denaturation electrophoresis

1. Prepare gel running buffer: 50 mM NaAc (DEPC treated) and 0.5 M EDTA (pH 8.0) in DEPC-treated H₂O.
2. Prepare 5x formaldehyde gel-running buffer (MOPS running buffer): 0.1 M MOPS (pH 7.0), 40 mM NaAc, 5 mM EDTA (pH 8.0).
   1. Dissolve 20.6 g of MOPS in 800 mL of DEPC treated 50 mM sodium acetate. Adjust the pH to 7.0 with 2 N NaOH. Add 10 mL of DEPC-treated 0.5 M EDTA (pH 8.0). Adjust the volume of the solution to 1 L with DEPC-treated water.
   2. Filter the solution through a 0.2 µm filter and keep it at room temperature away from light for the sake of sterilization. The buffer becomes yellow with time if it is exposed to light or is autoclaved. A straw-colored buffer works well, but darker ones do not¹⁷.
3. Prepare a 1.5% agarose gel¹⁸. For 50 mL, add 0.75 g of agarose and 31 mL of H₂O. Microwave the solution in the microwave for 1 min. Add 9 mL of formaldehyde and 10 mL of 5x MOPS running buffer.
4. Prepare the samples for the gel. Mix the following in a sterile microfuge tube:
   X µL RNA (up to 30 µg)
   2 µL of 5x gel-loading buffer
   10 µL of formamide
   4 µL of MOPS running buffer
   1 µL of 0.1 mg/mL EtBr
   3 µL of formaldehyde 5-x µL of DEPC-H₂O
5. Incubate the samples at 65 °C for 15 min and cool them on ice. Centrifuge for 5 s until all the fluid is deposited.
6. Pre-run the gel at 5 V/cm for 5 minutes.
7. Load the samples into the lanes of the gel immediately and then submerge the gel in 1x formaldehyde gel-running buffer. Run at 3-4 V/cm¹⁹.