Name: rapid and cost-effective rna extraction of rat pancreatic tissue
Uploaded: 2020-09-19T14:00:00
Description: Watch this Scientific Journal Video about Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue at JoVE.com

The present study was financially supported by Shiraz University of Medical Sciences (Grant No. 93-01-01-7178\03-07-2014). We thank Mr. Zomorodian and Mr. Rostami at the Department of e-Learning in Medical Sciences, Virtual School and Center of Excellence in e-Learning, Shiraz University of Medical Sciences for editing the video.

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&#8211;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 &#176;C and use RNA extraction reagent solution to maintain the integrity of RNA.
1. Removal of the rat pancreatic tissue
<ol>
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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 &#176;C to inactiva...

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-exchange1,2.
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 construction3,4.
RNA becomes rather unstable after being kept for a long time. Long mRNA fragments over 10 kb are particularly susceptible to degradation5,6. 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 practically7,8.
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 ineffective7,8,9,10,11,12,13,14.
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.

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

rapid and cost-effective rna extraction of rat pancreatic tissue

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 &#176;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.

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 ...

Watch this Scientific Journal Video about Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue at JoVE.com

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

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.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

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