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>
	<li>Prepare the operating room and place all required materials under the hood.</li>
	<li>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 &#176;C to inactivate RNases15. Sterilize the surface of the surgery place with 70% alcohol under the hood.</li>
	<li>Inject ketamine/xylazine intraperitoneally using an insulin syringe [80/8 mg/kg]. Check the depth of anesthesia by pinching the rat&#8217;s toes for a lack of response.</li>
	<li>Add 1 mL of RNA stabilization reagent in the proper microtube (2 mL) to inhibit activation of endonucleases in excised tissues.</li>
	<li>Immediately place the rat on the surgical board (head away from the surgeon) for a mid-line incision.</li>
	<li>Sterilize the entire abdominal surface with 70% alcohol and remove rat hair using a hair clipper with #40 blades.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the microtube in a nitrogen tank to freeze immediately. Keep the microtube in a -80 &#176;C freezer for 24 h16.</li>
	<li>After 24 h, transfer the tiny pieces of pancreatic tissues to an ice container.</li>
	<li>Inject potassium chloride (KCl) intracardially for euthanasia of rats after surgery.</li>
</ol>
2. RNA extraction
<ol>
	<li>Sterilize the surface under the hood with 70% alcohol.</li>
	<li>Put 1 mL of the RNA extraction reagent, which contains guanidinium thiocyanate to inhibit RNase, in the sterile microtube.</li>
	<li>Transfer the separated pancreatic tissue to the microtube. Transfer the microtube to the liquid nitrogen to freeze immediately.</li>
	<li>Homogenize the tissue with the micro tip probe sonicator at 4 &#176;C set to level 20 for 60 s on and 5 s off.</li>
	<li>Incubate each homogenized sample for 5 min at 4 &#176;C using crushed ice to allow the nucleoprotein complexes to be completely dissociated.</li>
	<li>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.</li>
	<li>Incubate the tube for 15 min on the crushed ice (4 &#176;C) to separate the reagent in three phases.</li>
	<li>Run the centrifuge at 12,000 x g for 15 min at +2 to +8 &#176;C. Transfer the colorless aqueous phase to a new microtube.</li>
	<li>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.</li>
	<li>Incubate the sample for 5-10 min on a cold box (4 &#176;C) to allow RNA precipitation.</li>
	<li>Run the centrifuge at 12,000 x g for 15 min at +2 to +8 &#176;C. Discard the supernatant.</li>
	<li>Enrich each of the centrifuge tubes with 1 mL of 75% ethanol.</li>
	<li>Wash the RNA pellet by inverting the tube at least 3 times.</li>
	<li>Run the centrifuge at 7,500 x g at +2 to +8 &#176;C for 5 min. Discard the supernatant to remove the excess ethanol from the RNA pellet by air-drying.</li>
	<li>Re-suspend the RNA pellet in diethylpyrocarbonate (DEPC)-treated RNase-free water.</li>
	<li>Pass the solution through a pipette tip several times to dissolve the RNA pellet. Then, incubate the solution for 10-15 min at +65 &#176;C.</li>
</ol>
3. Evaluating RNA Integrity with denaturation electrophoresis
<ol>
	<li>Prepare gel running buffer: 50 mM NaAc (DEPC treated) and 0.5 M EDTA (pH 8.0) in DEPC-treated H2O.</li>
	<li>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).
	<ol>
		<li>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.</li>
		<li>Filter the solution through a 0.2 &#181;m 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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Prepare the samples for the gel. Mix the following in a sterile microfuge tube: 
	X &#181;L RNA (up to 30 &#181;g) 
	2 &#181;L of 5x gel-loading buffer 
	10 &#181;L of formamide 
	4 &#181;L of MOPS running buffer 
	1 &#181;L of 0.1 mg/mL EtBr 
	3 &#181;L of formaldehyde 5-x &#181;L of DEPC-H2O</li>
	<li>Incubate the samples at 65 &#176;C for 15 min and cool them on ice. Centrifuge for 5 s until all the fluid is deposited.</li>
	<li>Pre-run the gel at 5 V/cm for 5 minutes.</li>
	<li>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.</li>
</ol>

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R., Sambrook, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+win+the+battle+with+RNase.">How to win the battle with RNase.</a> Cold Spring Harbor Protocols. (2), 101857 (2019).</li><li>Li, D. -. S., Yuan, Y. -. H., Tu, H. -. J., Dai, L. -. j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+islet+isolation+from+mouse+pancreas.">A protocol for islet isolation from mouse pancreas.</a> Nature Protocols. 4 (11), 1649 (2009).</li><li>Armstrong, J. A., Schulz, J. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis.">Agarose gel electrophoresis.</a> Current Protocol: Essential Laboratory Techniques. (1), 1-20 (2008).</li><li>Aranda, P. S., LaJoie, D. 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None declared.

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 ...

rapid-and-cost-effective-rna-extraction-of-rat-pancreatic-tissue

Research

JoVE Journal

Biochemistry

9.2K Views.  Shiraz University of Medical Sciences. Hallo allemaal. We hebben ontworpen, een eenvoudige, snelle in klasse beschrijvende methode voor efficiënte RNA extractie uit alvleesklierweefsel. Het belangrijkste voordeel van deze methode is een geoptimaliseerde procedure om RNA te extraheren uit een minimale hoeveelheid weefsel als gevolg van autolyse. Hallo weer. Demonstreren van de procedure zal worden gedaan door Sanaz Dastghaib, dat is mijn promovendus in ons lab op biochemie afdeling van Shiraz University of Medical Sciences.First b...

Video: Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Genetics

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Biology

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Medicine

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Developmental Biology

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Immunology and Infection

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

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