本研究由设拉子医科大学提供财政支持（第93-01-01-7178-03-07-2014号格兰特）。我们感谢设拉子医科大学医学电子学习系、虚拟学校和电子学习卓越中心的佐莫罗迪安和罗斯塔米先生编辑了视频。

这项研究 的 伦理 批准得到了 设拉子医科大学（批准编号：93-01-01-7178=03-07-2014）。
注：使用重达250克的雄性斯普拉格-道利大鼠。将含有胰腺组织片片的小瓶浸入RNA稳定试剂中，在-80°C处使用RNA提取试剂溶液保持RNA的完整性。
1. 切除大鼠胰腺组织
<ol><li>准备手术室，将所有所需材料放在机罩下。</li>
	<li>在240°C下，将所有手术器械（修整钳子、棕色-Adson钳子、虹膜剪刀和利弗剪刀）消毒至少4小时，以灭活RNases15。用70%的酒精在手术场所的表面进行消毒。</li>
	<li>使用胰岛素注射器[80/8毫克/千克]在单酮内注射氯胺酮。。通过捏住大鼠的手指趾，检查麻醉深度，以寻找缺乏反应。</li>
	<li>在适当的微管（2 mL）中加入1 mL的RNA稳定试剂，以抑制切除组织内核糖的活化。</li>
	<li>立即将大鼠放在手术板上（头部远离外科医生），进行中线切口。</li>
	<li>用 70% 的酒精消毒整个腹部表面，并使用带一根带刀片的剪子#40去除#40毛。</li>
	<li>做一个 V 形切口，用梳妆钳、棕色 - Adson 钳子、虹膜剪刀和利弗剪刀将腹部从区域打开到前腿。</li>
	<li>翻转腹部器官到左侧暴露胰腺。仔细找到在腹腔中扩散的胰腺组织。找到脾脏下的区域，找到胰腺，而不会与脂肪组织混淆。</li>
	<li>立即从腹腔中取出20-30毫克胰腺，不到2分钟。将去除的组织快速放入2 mL微管中，用无菌切割机切割，用RNA稳定试剂穿透分离的组织。</li>
	<li>将微管放在氮气罐中，立即冻结。将微管放在-80°C冷冻室中24小时16分。</li>
	<li>24小时后，将胰腺小片转移到冰容器中。</li>
	<li>在手术后注射氯化钾（KCl）供大鼠安乐死。</li>
</ol>2. RNA提取
<ol><li>用 70% 的酒精对发动机罩下的表面进行消毒。</li>
	<li>将1 mL的RNA萃取试剂，其中含有硫化铀，以抑制RNase，在无菌微管。</li>
	<li>将分离的胰腺组织转移到微管。将微管转移到液氮中立即冻结。</li>
	<li>在 4 °C 下，与微型尖端探针声波器将组织均质化，设置为 20 级，60 秒，关闭 5 秒。</li>
	<li>使用碎冰在4°C下孵育每个均质样品5分钟，使核蛋白复合物完全分离。</li>
	<li>每1mLRNA提取试剂，用0.2 mL的氯仿丰富样品。用力盖住管子，用力摇动管子15 s。</li>
	<li>在碎冰（4°C）上孵育管子15分钟，将试剂分三个阶段分离。</li>
	<li>在 +2 至 +8 °C 下以 12，000 x g 运行离心机 15 分钟。 将无色水相转移到新的微管。</li>
	<li>在水相中加入0.5 mL的100%异丙醇。关闭管，然后反转至少3次，彻底混合RNA。</li>
	<li>在冷盒（4°C）上孵育样品5-10分钟，允许RNA沉淀。</li>
	<li>在 +2 至 +8 °C 下以 12，000 x g 运行离心机 15 分钟。 丢弃上一杯。</li>
	<li>使用 1 mL 的 75% 乙醇丰富每个离心管。</li>
	<li>将管子反转3次，清洗RNA颗粒。</li>
	<li>在 +2 至 +8 °C 下以 7，500 x g 运行离心机 5 分钟。丢弃上一液，通过风干从RNA颗粒中去除多余的乙醇。</li>
	<li>将RNA颗粒重新悬浮在二乙基碳酸盐（DEPC）处理的无RNase水中。</li>
	<li>通过移液器尖端多次传递溶液以溶解RNA颗粒。然后，在 +65 °C 下孵育溶液 10-15 分钟。</li>
</ol>3. 用变性电泳评估RNA完整性
<ol><li>准备凝胶运行缓冲液：50 mM NaAc（DEPC处理）和0.5 M EDTA （pH 8.0）在DEPC处理H2O。</li>
	<li>准备 5x 甲醛凝胶运行缓冲液（MOPS 运行缓冲液）：0.1 M MOPS （pH 7.0）、40 mM NaAc、5 mM EDTA （pH 8.0）。<ol>
<li>溶解20.6克MS在800 mL的DEPC处理50mM醋酸钠。使用 2 N NaOH 将 pH 调整为 7.0。添加 10 mL 的 DEPC 处理 0.5 M EDTA （pH 8.0）。使用 DEPC 处理的水将溶液的体积调整为 1 L。</li>
		<li>通过 0.2 μm 过滤器过滤溶液，并在室温下远离光线，以便进行灭菌。如果缓冲区暴露在光线下或被自动遮挡，则缓冲区会随着时间而变黄。稻草色的缓冲器效果很好，但较暗的缓冲器不能有效。</li>
	</ol>
</li>
	<li>准备1.5%的加糖凝胶18。对于 50 mL，加入 0.75 g 的 agarose 和 31 mL 的 H2O. 微波在微波炉中加热 1 分钟。加入9 mL甲醛和10 mL的5x MOPS运行缓冲液。</li>
	<li>为凝胶准备样品。在无菌微混管中混合以下： X μL RNA （高达 30 μg） 2 μL 的 5x 凝胶加载缓冲液 10 μL 甲酰胺 4 μL 的 MOPS 运行缓冲器 1 μL 0.1毫克/mL EtBr 3 μL 甲醛 5-x μL 的 DEPC-H2O</li>
	<li>在65°C下孵育样品15分钟，并在冰上冷却。离心5 s，直到所有液体沉积。</li>
	<li>以 5 V/cm 的速度预运行凝胶 5 分钟。</li>
	<li>立即将样品装入凝胶的通道，然后将凝胶淹没在 1x 甲醛凝胶运行缓冲液中。运行在3-4 V/厘米19.</li>
</ol>

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在分子生物学中，获得高质量的RNA至关重要。核糖核酸酶在细胞和组织中的存在会迅速降解RNA，使提取变得复杂。RNases 是稳定的酶功能，没有任何共同因素。少量的RNase足以破坏RNA。当大鼠胰腺组织从腹腔中取出时，需要用强洗涤剂对手术器械进行消毒，彻底冲洗，并在240°C下将其放入烤箱至少4小时，在手术前灭活RNases。鉴于RNase水平在胰腺中非常高，手术地点用NaOH和轻度漂白剂进行灭菌，以停...

结构基因数据可以通过基因表达转录到功能产品。RNA分析用于发现不同条件下基因表达的差异。提取核酸的方法有很多，如下：硫化核酸，通过苯酚-氯仿提取，纤维素基色谱，二氧化硅基提取，和核子交换1，2。,2
正确检测基因表达受从组织分离的RNA的完整性影响;因此，在进行进一步测试之前，评估从组织分离的RNA的完整性至关重要，因为对低质量RNA的补充分子测试可能会危及诊断应用结果。因此，具有不同诊断应用的分子生物学测试需要高完整性RNA：定量RT-PCR、微阵列、核糖核酸酶保护测定、北方印迹分析、RNA映射和cDNA库结构3、4。,4
长期保存后，RNA变得相当不稳定。超过10 kb的长mRNA片段特别容易降解5,5，6。因此，研究人员必须考虑影响纯化RNA完整性的各种因素。RNA的纯度必须受到保护，防止RNA、蛋白质、基因组DNA和酶抑制剂污染。此外，RNA 与 UV （260/280） 的最佳和可接受的吸收比必须在 1.8-2.0 范围内，与电泳的碎片最小。最近开发的实验室技术使科学家能够更实际地评估分子分析样本的完整性,。
从胰腺组织中提取未受损的RNA比从其他类型的组织中提取未受损的RNA要困难得多，因为核糖核糖核酸（RNases）的量很高。然而，现有的提取方法，即胰腺组织从腹腔快速弹出和低温均质，以,阻碍RNases，已被证明是无效的7，8，9，10，11，12，13，14。89,10,11,12,13,14
本比较实验研究的目的是修改和比较现有方法，以确定最有效的方法。为此，对RNA提取的各种方案进行了修改和比较。它特别旨在确定最昂贵的方法，需要最低数量的胰腺组织。

<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

无论提取方法如何，组织和细胞系的优化RNA提取分四个阶段进行：1） 均质化，2） RNA中蛋白质的有效变性，3） 核糖核糖核素灭活，以及 4） 去除DNA、蛋白质和碳水化合物中的污染。然而，当组织中有高水平的RNA时，保持RNA的完整性是非常费力的。自发的自解使得在不损害胰腺组织的情况下从胰腺组织中提取RNA变得非常困难。因此，在提取过程中需要一种实用的RNA提取方法来维持胰腺组织的完整性。通过在不到2分钟内获得20-30毫克大鼠胰腺组织并提取RNA，对现有方案进行了实验和比较研究。结果通过电泳评估。实验进行了三次，以概括结果。当RNA提取试剂用作试剂时，在-80°C下将胰腺组织浸入RNA稳定试剂中24小时，产生高完整性RNA。所得结果与具有自旋柱结合的商业套件所得结果相当。

根据常规且经过修改的不带RNA稳定试剂的手术方案评估RNA提取试剂中RNA的完整性 使用RNA提取试剂从常规手术方案提取RNA后，观察到不可接受的波段。车道1显示来自肝脏的RNA作为对照。车道2显示了从常规手术方案获得的28S/18S rRNA波段的退化状态。当胰腺组织的数量减少到50毫克（车道3）或20-30毫克（车道4），并在没有RNA稳定试剂的情况下立即进行手术（修改方案）时，RNA分离不如?...

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

分离的RNA的纯度和完整性是RNA依赖性测定中的重要一步。在这里，我们提出了一个实用、快速和廉价的方法，从少量未受损的胰腺组织中提取RNA。

大鼠胰腺组织快速、经济高效的RNA提取

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

大家好。我们设计了一种简单、快速的类描述方法，用于从胰腺组织中高效提取RNA。这种方法的主要优点是优化了从最小组织中提取RNA的优化程序，由于自解。再次你好。演示程序将由Sanaz Dastghaib完成，这是我的博士生，在我们的实验室在设拉子医科大学生物化学系。灭活RNase在烤箱中消毒所有的手术仪器。每公斤注射氯胺酮-锡氨酸80/8毫克。将1个cc.RNA稳定试剂加入适当的微管，在现有的组织中活化内核糖。立即将老鼠放在手术板上。给外科医生做中线切口。然后，用70%的酒精消毒所有腹部表面，去除老鼠的头发。切皮肤做手术。重塑切口，将腹部从区域打开至前腿。翻转腹部器官到左侧，露出胰腺。仔细找到在腹腔中传播的胰腺组织。找到显示下的区域，找出胰腺，而不会与脂肪组织混淆。在不到两分钟内立即从腹腔中取出20至30毫克胰腺。将去除的组织迅速放入微管中，用刀刀切割，将RNA稳定试剂穿透分离组织。将微管放在氮气罐中，立即冻结。他们所有的微管应该保存在 80

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

Research

JoVE Journal

Biochemistry

9.1K 次观看.  Shiraz University of Medical Sciences. 大家好。我们设计了一种简单、快速的类描述方法，用于从胰腺组织中高效提取RNA。这种方法的主要优点是优化了从最小组织中提取RNA的优化程序，由于自解。再次你好。演示程序将由Sanaz Dastghaib完成，这是我的博士生，在我们的实验室在设拉子医科大学生物化学系。灭活RNase在烤箱中消毒所有的手术仪器。每公斤注射氯胺酮-锡氨酸80/8毫克。将1个cc.RNA稳定试剂加入适当的微...

视频: 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.

从人体二尖瓣提取蛋白质的优化方案

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

科研

生物化学

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.

小鼠脂肪组织的采集和 RNA 分析处理

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.

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

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.

HEK 细胞 Mitoribosome 的快速分离

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.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

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.

台湾绿蜂胶的提取与分析

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.

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

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.

极快速和特殊的代谢标签的RNA在Vivo与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 ...

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.

神经节苷脂提取、纯化和分析

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.