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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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 ...

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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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Research

JoVE Journal

Biochemistry

9.1K Views.  Shiraz University of Medical Sciences. 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.

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

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Extraction and Analysis of Taiwanese Green Propolis

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.