Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Summary

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Abstract

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 °C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

Introduction

Structural gene data can be transcribed to a functional product through gene expression. RNA analysis is used to discover differences in gene expression across different conditions. There are a number of methods to extract nucleic acids as follows: guanidinium thiocyanate, extraction via phenol-chloroform, cellulose-based chromatography, extraction by silica matrices, and anion-exchange^1,2.

Proper detection of gene expression is influenced by the integrity of RNA isolated from tissues; therefore, it is vital to evaluate the integrity of RNA isolated from tissues before further tests are carried out because complementary molecular tests on low-quality RNA may jeopardize diagnostic application results. Thus, high integrity RNA is needed for molecular biological tests with different diagnostic applications: quantitative RT-PCR, micro-arrays, ribonuclease protection assay, northern blot analysis, RNA mapping, and cDNA library construction^3,4.

RNA becomes rather unstable after being kept for a long time. Long mRNA fragments over 10 kb are particularly susceptible to degradation^5,6. Thus, researchers must consider various factors that influence the integrity of purified RNA. The purity of RNA must be protected against RNases, proteins, genomic DNA, and enzymatic inhibitor contamination. In addition, the best and acceptable absorption ratio of RNA to UV (260/280) must be within the range of 1.8-2.0 with minimum fragmentation over electrophoresis. Recently developed laboratory techniques have enabled scientists to evaluate the integrity of molecular analysis sample more practically^7,8.

It is much more difficult to extract undamaged RNA from pancreatic tissue than other types of tissues because of the high quantity of ribonucleases (RNases). However, existing extraction methods, namely the rapid ejection of the pancreatic tissue from the abdominal cavity and homogenization at low temperatures to impede RNases, have proven ineffective^{7,8,9,10,11,12,13,14}.

The purpose of the present comparative experimental study is to modify and compare existing methods to determine the most efficient methods. To that end, various protocols of RNA extraction were modified and compared. It was specifically aimed at determining the least expensive method requiring a minimum amount of pancreatic tissue.

Protocol

Ethical approval for this study was obtained from Shiraz University of Medical Sciences (Approval number: 93-01-01-7178\03-07-2014).

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.

Results

Evaluation of the integrity of RNA in the RNA extraction reagent according to a routine and modified surgical protocol without RNA stabilization reagent
Unacceptable bands were observed after the extraction of RNA with the RNA extraction reagent from a routine surgical protocol. Lane 1 shows RNA from the liver as a control. Lane 2 shows the degraded status of 28S/18S rRNA bands in total RNA obtained from a routine surgical protocol. When the quantity of pancreatic tissue was reduced to 50 mg (lane ...

Discussion

In molecular biology it is vital to obtain high-quality RNA. The presence of the ribonuclease enzymes in cells and tissues quickly degrades RNA and makes the extraction complex. RNases are stable enzymes functioning without any co-factors. Small amounts of RNase are adequate to destroy RNA. When the rat pancreatic tissue is removed from the abdominal cavity, it is necessary to disinfect the surgical instruments by strong detergents, rinse them thoroughly and put them in an oven for at least 4 h at 240 °C to inactiva...

Materials

Name	Company	Catalog Number	Comments
Agarose	Merck	116801	Germany
Atoclave	Teb Zaim		Iran
Centrifuge	Sigma		Germany
Chloroform	Merck	107024	Germany
Diethylpyrocarbonate (DEPC)-treated water	Sigma		Germany
EDTA	sigma	60-00-4	Germany
Electrophoresis tank	Payapajoohesh		Iran
Eppendorf microTube	Extragene		Taiwan
EtBr	sigma	E 8751	Germany
Ethanol	Merck	81870	Germany
Falcon Tube	Extragene		Taiwan
Formaldehyde	Merck	344198	Germany
Formamide	Merck	344206	Germany
Homogenizer-sunicator	Microson XL 2000		USA
Isopropanol	sigma	19516	Germany
Ketamine hydrochloride	sigma	1867-66-9	Germany
Laminar Flow Hood	Jal Tajhiz		Iran
Mgnetic stirrer	Labrotechnik		USA
Microcentrifuge	Eppendorf		Germany
Micropipette Tips	Extragene		Taiwan
MOPS	sigma	85022106	Germany
Na AC	Merck	567422	Germany
NaOH	Merck	109137	Germany
Oven	Teb Zaim		Iran
PH meter	Knick		Germany
RNA Later/RNA stabilization reagent	Qiagen	76104	USA
Surgical instrument	Agn Thos		German made
Syringes	AvaPezeshk		Iran
TriPure reagent/RNA extraction reagent	Roche	11667157001	USA
Vortex	Labinco		Netherland
Water bath	Memmert		Germany
zylazine	sigma	7361-61-7	Germany