Name: dna extraction and comparison between old and fresh necrophilic fly samples
Uploaded: 2024-05-03T14:10:00.000+00:00
Duration: 6 min 33 s
Description: Read this Scientific Article Protocol about DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples at JoVE.com

这项工作得到了中国国家自然科学基金 （82060341,81560304） 和海南省院士创新平台科学研究项目 （YSPTZX202134） 的支持。

注意：本方案共使用了 5 个 C. megacephala 样品 - 3 个新鲜样品（新鲜 1、新鲜 2 和新鲜 3），1 个样品在酒精中储存 2 年（Old 1），1 个样品在干燥密封储存中保存 2 年仅避光（Old 2）。样品必须根据实验要求进行标记。
1. 一般样品储存和制备
<ol><li>样品选择和制备 注意：使用乙酸乙酯处决坏死蝇时，请务必在通风橱中工作。称重前，从果蝇表面去除液体。<ol><li>新鲜样品：用乙酸乙酯杀死后，将收集的三个苍蝇样品放入气瓶中。称量样品并将其命名为 Fresh 1、Fresh 2 和 Fresh 3，并立即在实验中使用。 注：该方案中的新鲜果蝇样品质量为 56.8 mg、49.3 mg 和 52.1 mg。</li>
		<li>旧样品 1：取出被乙酸乙酯杀死的果蝇样品，并在充满酒精的离心管中密闭保存 2 年，在室温下避光保存。用滤纸吸附表面液体后称取样品;称之为 Old 1。 注意：在该方案中，Old 1 的质量为 42.3 mg。</li>
		<li>旧样品 2：取出被乙酸乙酯杀死的果蝇样品，并在离心管中密闭保存 2 年，在室温下避光保存。称重并称其为 Old 2。</li>
	</ol></li>
	<li>提取前的准备<ol><li>加入少量液氮冷却不锈钢容器;等到所有液氮都蒸发完毕。</li>
		<li>将苍蝇样本放入容器中;缓慢倒入液氮，以防止液氮飞溅并影响昆虫样品。</li>
		<li>将样品冷冻在液氮中。液氮蒸发后取出样品。</li>
		<li>单独分离胸部，放入 2 mL 离心管中，切成尽可能小的切片。 注：将样品切成小块，使其可以完全裂解，并且可以大量释放 DNA。</li>
		<li>对每只果蝇重复上述步骤，单独冷冻，然后转移到新的离心管中，以防止 DNA 交叉污染。</li>
	</ol></li>
</ol>2. DNA 提取
注：所有离心步骤必须在室温 （15-25 °C） 下在微量离心机中进行。所有步骤都必须严格遵守无菌原则并避免交叉污染。
<ol><li>组织裂解<ol><li>将 180 μL 组织裂解缓冲液添加到含有夹住样品的 2 mL 离心管中。</li>
		<li>加入 20 μL 蛋白酶 K。以 3 000 rpm 涡旋 10 秒充分混合，并在 56 °C 下孵育直至昆虫组织完全裂解。 注意：如果组织未完全消化，则应添加更多蛋白酶 K。裂解通常在 1-3 小时内完成，也可延长裂解时间过夜。</li>
	</ol></li>
	<li>DNA 沉淀<ol><li>以 3,000 rpm 的速度涡旋 15 秒。向样品中加入 200 μL 裂解缓冲液，并以 3,000 rpm 涡旋 15 秒充分混合。</li>
		<li>加入 200 μL 乙醇 （96-100%），并以 3,000 rpm 涡旋 15 秒，再次充分混合。 注：裂解缓冲液和乙醇可以一步预添加在一起，以节省时间。加入裂解缓冲液和乙醇后可能会形成白色沉淀。</li>
	</ol></li>
	<li>将混合物移液到放置在 2 mL 收集管中的 DNA 吸附柱中。以 6,000 × g 离心 1 分钟。丢弃流通管和收集管。</li>
	<li>将上一步的 DNA 吸附柱放入新的 2 mL 收集管中，加入 500 μL 蛋白质去除缓冲液，并以 6,000 × g 离心 1 分钟。丢弃流通管和收集管。</li>
	<li>将 DNA 吸附柱转移到新的 2 mL 收集管中，加入 500 μL 脱盐缓冲液，并以 20,000 × g 离心 3 分钟以干燥 DNA 吸附柱膜。丢弃流通管和收集管。 注：此离心步骤可确保在下一次洗脱之前去除任何残留的乙醇。如果有任何乙醇残留，再次以 20,000 × g 离心 1 分钟。</li>
	<li>将 DNA 吸附柱置于干净的 2 mL 微量离心管中，直接将 100 μL DNA 洗脱缓冲液吸至柱膜上，室温孵育 1 分钟，然后以 6,000 × g 离心 1 分钟洗脱。 注：用 100 μL（而不是 200 μL）洗脱会增加洗脱液中的最终 DNA 浓度，但也会降低 DNA 总产量。为了最大限度地提高 DNA 产量，请按照步骤 2.8 中的说明重复洗脱一次。</li>
	<li>将 DNA 样品储存在 4 °C（长达 1 周）或 -80 °C 下进行长期储存。</li>
</ol>3. DNA 浓度检测
<ol><li>通过酶标仪确定 OD 260 和 OD 280 的值，以比较 DNA 纯度和核酸浓度。对每个样品重复 3 次。</li>
	<li>使用其配套试剂盒通过荧光计测定提取的 DNA 的浓度，每个样品重复 3 次。<ol><li>取 190 μL 工作溶液放入两个 0.5 mL 离心管中，取标准 #1 和标准 #2 各 10 μL，加入工作溶液中，避光放置不少于 15 分钟。 注：标准品 #1 和标准品 #2 针对一定浓度的 DNA 进行了精确校准。应准确配置标准溶液，以确保形成准确的标准曲线。标准 #1 是 0.2 ng/μL 双链 DNA （dsDNA） 样品;标准 #2 为 2,000 ng/μL。工作溶液是一种与 dsDNA 特异性结合的染料。</li>
		<li>取 2 μL 检测 DNA，在 0.5 mL 离心管中与 198 μL 工作溶液混合，避光不少于 15 分钟。 注：如果 DNA 浓度低，则添加 5 μL 测试 DNA 和 195 μL 工作溶液。</li>
		<li>15 分钟后，打开仪器并选择 dsDNA 浓度测量 |长距离。首先，测试标准 #1，然后测试标准 #2 以获得标准曲线。 注意：标准溶液必须在配置后的同一天使用;放置时间过长会导致标准曲线出现较大的误差。</li>
		<li>将待测样品逐一放入检测孔中，将 DNA 加标体积调节至 2 μL，重复测量 3 次，取平均值，记录。</li>
	</ol></li>
</ol>4. PCR 和电泳检测
注：取长度为 278 bp 的线粒体 CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;"> 基因序列，用正向引物 C1-J-2495：CAGCTACTTTATGAGCTTTAGG，反向引物 C1-N-2800：CATTTCAAGCTGTGTAAGCATC8 进行 PCR 扩增，然后进行 2% 琼脂糖凝胶电泳，验证扩增效果和扩增产物的长度。
<ol><li>通过添加 10 μL 2x Taq 混合物、1 μL 每种引物、5 μL ddH2O 和 3 μL 模板 DNA，构建 20 μL PCR 扩增系统;在离心机中充分混合 10 秒。</li>
	<li>使用以下PCR循环参数：预变性95°C，10分钟;变性 95 °C 持续 30 秒，退火 48 °C 持续 30 秒，延伸 72 °C 持续 45 秒，共 40 个循环;最终延伸 72 °C 10 分钟。</li>
	<li>将 PCR 后的产物进行 2% 琼脂糖凝胶电泳，电泳后，将它们放在凝胶成像系统上进行光分析。</li>
</ol>

评估新鲜和老嗜坏死果蝇样品中的 DNA 纯度和浓度

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作者声明他们没有相互竞争的经济利益。

DNA 提取是嗜坏死果蝇分子鉴定中最关键的环节，其提取方法和提取的 DNA 质量直接影响后续检测。在本实验中，我们使用普通的血液试剂盒成功地从嗜坏死的果蝇中提取了 DNA，而无需购买特殊的昆虫 DNA 提取试剂盒，这使得昆虫 DNA 提取更容易完成。
果蝇表面富含几丁质，尤其是在幼虫和蛹期，表皮几丁质可达 60-70%，这使得嗜坏死果蝇的 DNA 提取变得困难9。在提取过程中，我们发现，如果样品是湿的，尽管我们已经充分剪切了样品，但当使用蛋白酶 K 进行消化时，样品仍呈凝胶状。这可以防止添加的液体通过吸附塔返回。为了解决这个问题，我们首先使用液氮快速冷冻样品，使果蝇变脆且易于研磨，并且易于完全消化。如果液氮无法研磨且 DNA 提取效率仍有待提高，则可以将果蝇样品放置在通风处约 48 小时。样品在充分剪切并随后提取 DNA 之前，可以让样品干燥。
考虑到老样本的 DNA 降解是一个更大的问题，由于胸部 DNA 含量高，本实验选择苍蝇胸部样本进行 DNA 提取。然而，胸部的使用对果蝇的形态完整性造成了太大的破坏，无法完全保存后续证据和形态学鉴定的需要。当胸部腐烂时，样本很难提取。如果样本是新鲜的，则最好提取足部和翅膀的 DNA 。足和翅对称分布和双侧分布，从一侧提取组织后可保留更完整的形态学特征。然而，从翅膀和足中提取的 DNA 浓度相对较低，这对后续研究来说是有风险的。此外，样本量小，容易丢失10.
当使用 DNA 提取试剂盒进行 DNA 提取时，除旧样品 2 外，酶标仪测得的 OD260/OD280 值均大于 2。首先，可以确定蛋白质被更彻底地去除，但可能存在 RNA 污染、由于重复离心而导致的 DNA 双链断裂或旧样品中的 DNA 降解11。其次，它也可能表明酒精会延长 RNA 的保存时间。
DNA 浓度是根据酶标仪的 OD260 值计算的，而 Qubit 荧光计首先用于建立标准曲线，然后测量吸光度值以计算 DNA 浓度。两种方法都可以实现痕量 DNA 浓度的测量，但各有优缺点：酶标仪应用 OD260 来计算 DNA 浓度，但其计算方法也是一种估计。如果样品中混有大量的核苷酸或蛋白质等能吸收紫外光的物质，光度误差会更大，所以我们应该尽量事先去除它们。
酶标仪和紫外分光光度计使用紫外吸光度进行检测，无法区分 DNA、RNA、降解核酸、游离核苷酸等杂质，而且吸光度受其他物质的污染也很大，使其读数相对不可靠。在加标样品（通常以液滴为单位）时，样品直接加标，无需稀释或处理，从而可以同时测量多个样品，并根据 OD260/OD280 测量值直接计算 DNA 纯度。另一方面，Qubit 荧光计旨在通过应用两种已知浓度的标准溶液来建立标准曲线，并应用与双链 DNA 特异性结合的荧光染料的特性，从而仅测量双链 DNA 的浓度。然而，每个样品都需要单独预处理，然后单独测量，这对于批量样品应用来说很麻烦，并且无法测量提取的 DNA 的纯度。在两者的具体应用中，可根据自身需要选择进行DNA质量检测12、13、14。
新鲜样品的 DNA 浓度高于置于酒精中的旧样品的 DNA 浓度，也高于未经处理的旧样品的 DNA 浓度，无论 DNA 浓度是通过酶标仪还是 Qubit 荧光计测量的。对于长期储存，样品可以浸入酒精中以防止细菌和真菌的生长15,16。
不同的测量方法对未经任何处理的旧样品的 DNA 测量影响较小，可能是因为旧样品的降解程度更高，尤其是蛋白质和 RNA 的降解速度都可能比 DNA 快。相比之下，新鲜样品含有更多的蛋白质和 RNA，因此对结果的影响更大。如果只比较双链 DNA 浓度，我们应该使用 Qubit 荧光计的结果，我们的实验结果也在新旧样品中产生了显着的 DNA 浓度梯度。
DNA 提取后新鲜和旧样品的 DNA 含量可用于后续的分子生物学分析。与本实验一样，为线粒体 CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;"> 基因选择的引物仅为 278 bp，并且都可以被 DNA 成功扩增。反过来，线粒体 CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;"> 基因是果蝇中因其 DNA 条形码潜力而被识别的基因座，因此可以通过随后的测序分析来识别嗜坏死的果蝇物种17。通过为其他较短的 DNA 序列选择引物，可以成功实现保存时间较长的样品的分子生物学。值得注意的是，旧样品的电泳条带有两个位置不同且序列较短的条带，这可能是由于 DNA 降解和低退火温度引起的引物非特异性扩增。
然而，在这个实验中，为了满足重复实验的需要，长期保存的样本较少，而结果只需要有代表性的样本。因此，我们没有进行重复性测试，这可能对新旧样品的保存具有探索性意义，但不影响嗜坏死苍蝇样品的成功提取。
总之，使用血液 DNA 提取试剂盒也有助于提取嗜坏血果蝇 DNA。新旧样品都可以成功提取，所有提取的 DNA 都可用于后续分析。酒精也有利于果蝇 DNA 样品的长期保存。

<a href="https://app-jove-com.remotexs.ntu.edu.sg/t/6604">This corrects</a> the article <a href="https://dx-doi-org.remotexs.ntu.edu.sg/10.3791/66737">10.3791/66737</a>

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/66737">DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples</a>. The Authors section was updated.

Erratum: DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples

死亡时间的推论一直是司法实践中需要解决的关键和难点问题之一。在法医学实践中，对于刑事案件，确定验尸间隔对于推断犯罪时间、锁定嫌疑人、缩小侦查范围起着至关重要的作用。传统的推断死亡时间的方法主要基于早期的死后现象，通常只能用于推断 24 小时内的死亡时间。然而，死亡时间的长短不能由死后现象来确定。现在，法医科学界普遍认为，法医昆虫学有可能成为推断较长死亡时间的有效方法。
Necrophagous 昆虫以其以尸体为食的幼虫而得名。其中，每当有尸体出现时，成年嗜尸蝇就会第一个到达尸体并产卵或幼虫。幼虫以尸体组织为食，成熟为蛹，蛹长成成虫。嗜尸蝇由于其有规律的生命周期和地理分布，在法医学实践中发挥着巨大的作用，例如，扣除死亡时间并确定死亡地点 1,2。然而，在法医实践中使用嗜尸蝇的障碍是物种鉴定。形态学方法仍被用作嗜坏死果蝇物种鉴定的权威方法。然而，形态学物种鉴定需要高度的昆虫完整性。如果果蝇处于不同的发育阶段，或者由于环境选择引起的形态变化，这些都会使形态学检查更加困难。特别是，最常见于犯罪现场的蛹和蛹壳的形态几乎无法辨认。这一点，再加上形态学专家的稀缺，使物种的形态学鉴定变得非常困难。因此，出现了分子生物学方法对果蝇物种鉴定的应用，它降低了形态学鉴定的难度，并且独立于发育阶段 3,4,5,6,7。
基于分子生物学方法对嗜坏死果蝇进行物种鉴定的第一步是高效提取 DNA，而目前不常用用于昆虫 DNA 提取的专用试剂盒。如何使用普通血液试剂盒提取嗜坏死果蝇的 DNA 变得更具法医意义。在犯罪现场发现的嗜尸蝇通常被肢解或经历腐烂和 DNA 降解。果蝇样品在采集后不能立即用于 DNA 提取，或者由于证据保存要求，如果可能，需要保留完整的样品。基于上述要求，本研究采用基于蛋白酶K消化的血液DNA试剂盒，选取3个新鲜 的大头金丝菌（双翅目、鳞翅目） 样本（图1），1个大 头念珠 菌老样本在酒精中放置两年，1个巨 头念珠 菌样本在避光保存中放置两年，对5个样本各称重， 选择了昆虫胸部组织进行 DNA 提取。比较从新旧样品中提取的 DNA 的质量和纯度，使用位于线粒体 CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;"> 基因序列中的嗜坏苍蝇特异性引物对提取的 DNA 进行 PCR 扩增和电泳。

<table><tbody><tr><td>Buffer AE</td><td>Qiagen</td><td>172026832</td><td>DNA elution buffer</td></tr><tr><td>Buffer AL</td><td>Qiagen</td><td>172028374</td><td>lysis buffer</td></tr><tr><td>Buffer ATL</td><td>Qiagen</td><td>172028162</td><td>tissue lysis buffer</td></tr><tr><td>Buffer AW1</td><td>Qiagen</td><td>172028760</td><td>protein removal buffer</td></tr><tr><td>Buffer AW2</td><td>Qiagen</td><td>57203108</td><td>desalination buffer</td></tr><tr><td>C1-J-2495</td><td>Taihe Biotechnology</td><td>TW21109216</td><td>forword primer</td></tr><tr><td>C1-N-2800</td><td>Taihe Biotechnology</td><td>TW21109217</td><td>reverse primer</td></tr><tr><td>D2000 DNA ladder</td><td>Real-Times(Beijing) Biotechnology</td><td>RTM415</td><td>Measure the position of electrophoretic bands</td></tr><tr><td>DNeasy Mini spin column</td><td>Qiagen</td><td>166050343</td><td>DNA adsorption column</td></tr><tr><td>Dry Bath Incubator</td><td>Miulab</td><td>DKT200-2D</td><td>used for heating</td></tr><tr><td>MIX-30S Mini Mixer</td><td>Miulab</td><td>MUC881206</td><td>oscillatory action</td></tr><tr><td>Proteinase K</td><td>Qiagen</td><td>172026218</td><td>Inactivation of intracellular nucleases and other proteins</td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>Thermo Fisher Scientific</td><td>2321611188</td><td></td></tr><tr><td>Speed Micro-Centrifuge</td><td>Scilogex</td><td>9013001121</td><td>centrifuge</td></tr><tr><td>Standard#1</td><td>Thermo Fisher Scientific</td><td>2342797</td><td></td></tr><tr><td>Standard#2</td><td>Thermo Fisher Scientific</td><td>2342797</td><td></td></tr><tr><td>Tanon 3500R Gel Imager</td><td>Tanon</td><td>16T5553R-455</td><td>gel imaging</td></tr><tr><td>Taq Mix Pro</td><td>Monad</td><td>00007808-140534</td><td>PCR Mix</td></tr><tr><td>Taq Mix Pro</td><td>Monad</td><td>00007808-140534</td><td>PCR Mix</td></tr><tr><td>Thermo Cycler</td><td>Zhuhai Hema</td><td>VRB020A</td><td>ordinary PCR</td></tr><tr><td>Working Solution</td><td>Thermo Fisher Scientific</td><td>2342797</td><td></td></tr></tbody></table>

dna extraction and comparison between old and fresh necrophilic fly samples

总共选择了 5 个大 头金鸡 孢菌样品 - 3 个新鲜样品，1 个样品在酒精中储存 2 年，1 个样品在干燥密封储存中避光保存 2 年 - 以研究血液 DNA 提取试剂盒是否可以从嗜坏蝇中提取 DNA，并确定酒精是否可以延长嗜坏蝇 DNA 的保存时间。首先，使用血液 DNA 提取试剂盒从他们的胸部组织中提取 DNA。然后，使用酶标仪和荧光计检查 DNA 纯度和浓度。最后，使用位于线粒体 CO I 基因序列中的嗜坏苍蝇特异性引物对提取的 DNA 进行 PCR 扩增和电泳。结果表明，所有样品的 DNA 纯度均大于 2.0。观察到 DNA 浓度按以下顺序排列：新鲜样品>酒精保存的旧样品>未经处理的旧样品。PCR 扩增后所有样品均具有特异性电泳条带。综上所述，血液 DNA 提取试剂盒可用于成功提取嗜坏死果蝇的 DNA，且鲜果蝇样本的 DNA 浓度大于老果蝇样本。苍蝇可以长时间储存在酒精中。

我们使用酶标仪测量提取的 DNA 溶液的 OD260 和 OD280 值，然后获得 OD260/OD280 值以评估 DNA 的纯度。所有新鲜样品和旧样品 1 的 OD260/OD280 均大于 2。旧样品 2 的 OD260/OD280 的最大值为 2.187，尽管平均值为 1.753，并且由于其 OD280 和 OD280 的值都较小 （≤0.01），其三次测量波动很大（表 1）。根据获得的 OD260 值，我们估计了 DNA 的浓度。三种样品的 DNA 浓度依次为：新鲜样品>旧样品 1 >旧样品 2，使用酶标仪测量，但新鲜样品与旧样品之间的差异很小，仅为 ~10-20 ng/μL（表 1），而 Qubit 荧光计测量的 3 个新鲜样品的 DNA 浓度比酶标仪测量的 DNA 浓度高 125-130 ng/μL， 旧样品 1 的差异为 15 ng/μL，旧样品 2 的差异仅为 1.3 ng/μL。新鲜样品>旧样品 1 >旧样品 2 的 DNA 浓度（表 2）。
通过当前使用的提取方法提取的 DNA 包含样品中来自不同来源的所有 DNA 成分。这意味着 DNA 溶液不仅仅是果蝇基因组 DNA 的溶液。通过查阅文献选择特异性扩增果蝇线粒体 CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;"> 基因片段的引物，并通过 NCBI-Blast Primer 验证。结果表明，线粒体 CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;"> 基因的正向引物 C1-J-2495 和反向引物 C1-N-2800 是具有序列多态性的果蝇特异性引物，覆盖了许多果蝇物种。扩增结果未显示人类基因。它们是一对可靠的 DNA 生物标志物，用于嗜坏死果蝇的鉴定，并被选中用于验证本实验的结果。
所有不同样品均表现出明显的电泳条带，长度范围为 250 bp-400 bp。新鲜样品的条带比旧样品更亮，酒精放置的旧样品（图 2，Old1）比未处理的旧样品（图 2，Old2）更亮，但旧样品在不同位置也有两条条带。空白对照没有条带（图 2）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66737/66737fig01.jpg"> 图 1：不同角度的 Chrysomya megacephala 图像。 （A） 雄性;（B） 女性。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66737/66737fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66737/66737fig02.jpg"> 图 2：PCR 扩增产物的电泳图。 左起前三个条带是新鲜果蝇样品中 DNA 的 PCR 扩增产物。第四个电泳条带是保存在酒精中的老果蝇样品中 DNA 的 PCR 产物。第五个条带是来自老果蝇样本的 DNA 的 PCR 产物，没有任何特殊保存。第六个频段是空白控件;它是干净的，证明在 PCR 扩增过程中没有发生污染。最右侧是 DNA 分子量标准，我们标记了长度为 250 bp 和 400 bp 的 DNA 位置。所有样品的 PCR 产物的电泳条带均略高于 250 bp 和低于 400 bp 的梯形条带。该位置与 278 bp 靶基因的长度相匹配。肉眼更明显的是，代表新鲜样品的 DNA 条带比旧样品的 DNA 条带更亮。旧样本包含其他 DNA 条带，这些条带存在于强度较低、不同长度的非靶基因中。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66737/66737fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>样本</td>
			<td>新鲜1</td>
			<td>新鲜2</td>
			<td>新鲜3</td>
			<td>旧1</td>
			<td>旧2</td>
		</tr><tr><td>OD260/OD280</td>
			<td>2.354±0.036</td>
			<td>2,245±0.027 元</td>
			<td>2.060±0.038</td>
			<td>2.143±0.012</td>
			<td>1.753±0.421</td>
		</tr><tr><td>DNA（ng/μL）</td>
			<td>119.91±1.686</td>
			<td>116.084±4.965</td>
			<td>95.239±3.401</td>
			<td>86.845±6.14</td>
			<td>10.151±0.168</td>
		</tr></tbody></table>表 1：使用酶标仪测量 DNA 纯度和浓度。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>样本</td>
			<td>新鲜1</td>
			<td>新鲜2</td>
			<td>新鲜3</td>
			<td>旧1</td>
			<td>旧2</td>
		</tr><tr><td>DNA（ng/μL）</td>
			<td>243.333±1.528</td>
			<td>237.667±2.082</td>
			<td>226.000±2.000 元</td>
			<td>101.000±0.000 元</td>
			<td>11.433±0.058 元</td>
		</tr></tbody></table>表 2：用荧光计测量 DNA 浓度。

Read this Scientific Article Protocol about DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples at JoVE.com

作者聚焦：使用简单技术推进嗜坏死果蝇样品的 DNA 提取

本文介绍了使用改良的常用 DNA 提取试剂盒提取新鲜和旧嗜坏死果蝇 DNA 的方案。

新旧嗜坏死果蝇样品的 DNA 提取和比较

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry ...

dna-extraction-comparison-between-old-fresh-necrophilic-fly

Nanyang Technological University

Research

JoVE Journal

Medicine

DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples

423 次观看.  Hainan Medical University, Hainan Provincial Academician Workstation (tropical forensic medicine). 我们的颗粒旨在简单方便地提取果蝇 DNA，并比较嗜坏死果蝇新旧样本之间的 DNA 浓度。我们正在尝试研究血液 DNA 提取试剂盒是否能有效地从果蝇中提取 DNA，以及嗜坏死果蝇的新旧样本的 DNA 浓度是否有变化？我们提供了一种使用普通血液检测试剂盒提取果蝇 DNA 的方法，解决了昆虫提取试剂盒昂贵和稀缺的问题，为这些试剂盒的分子生物学分析铺平...

视频: 作者聚焦：使用简单技术推进嗜坏死果蝇样品的 DNA 提取

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

龟科害虫的大规模饲养与分子研究

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

科研

环境科学

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

神经科学

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

生物学

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

与神经退化有关的成人 果 蝇的可视化突触变性

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色<em&gt;果蝇</em&gt;组织<em&gt;原位</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

使用 Vivo 法戈西西亚赛评估成人嗜血杆菌黑色粒细胞的年龄特异性噬细胞能力

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

Circadian Entrainment of Drosophila Melanogaster

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this rhythm and to understand the resulting outputs, entrainment of model organisms to defined circadian time-points is required. Here we detail a procedure to entrain many Drosophila to a defined circadian rhythm. Furthermore, we detail post-entrainment steps to prepare samples for immunofluorescence, nucleic acid, or protein extraction-based analysis.

Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology.

果蝇黑色素的循环训练

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this ...

<meta charset="utf8"></head><body>果蝇眼缺陷的高分辨率成像和分析

A Rapid, Simple Workflow for Quantification of External Adult Drosophila Structures

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It is a simple structure of repeating unit eyes, termed ommatidia, that is used to characterize genetic interactions and gene functions. Mutations that affect eye architecture can be easily detected and analyzed; hence, this system is frequently used in under-resourced institutions. Further phenotypic analysis often includes a Scanning Electron Microscope (SEM) to generate high-magnification images suitable for quantitative analysis. However, SEMs are expensive and require costly reagents; sample preparation spans days; and, often, they need full-time staff for sample preparation and instrument maintenance. This limits their utility at under-resourced institutions or during budgetary austerity. In entomology, the use of high-resolution digital imaging technology is a common practice for the identification and characterization of species. This paper describes a method that combines strategies and allows for high-resolution digital imaging of adult Drosophila structures and quantitative analysis using the open software ImageJ. The workflow is a rapid and student-friendly alternative that remedies the limitations of underfunded and under-resourced research facilities with a cost-effective and rapid approach to quantitative phenotypic analysis.

<meta charset="utf8"></head><body>用于定量体外成体 果蝇 结构的快速、简单的工作流程

Here, we present a rapid, low-cost, workflow for high-resolution imaging of adult Drosophila eyes to quantify patterning and growth defects. We describe our protocol for sample preparation by point-mounting, high-resolution image acquisition, and image analysis.

用于定量体外成体 果蝇 结构的快速、简单的工作流程

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It ...

新旧嗜坏死果蝇样品的 DNA 提取和比较

副本

摘要

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此视频中的章节

龟科害虫的大规模饲养与分子研究

大众组织学量化在神经退行性变

成绩单和代谢产物的分离纯化

与神经退化有关的成人果蝇的可视化突触变性

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

使用 Vivo 法戈西西亚赛评估成人嗜血杆菌黑色粒细胞的年龄特异性噬细胞能力

果蝇黑色素的循环训练

用于定量体外成体果蝇结构的快速、简单的工作流程

用于定量体外成体果蝇结构的快速、简单的工作流程

新旧嗜坏死果蝇样品的 DNA 提取和比较

副本

摘要

探索更多视频

此视频中的章节

龟科害虫的大规模饲养与分子研究

大众组织学量化在神经退行性变

成绩单和代谢产物的分离纯化

与神经退化有关的成人 果 蝇的可视化突触变性

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

使用 Vivo 法戈西西亚赛评估成人嗜血杆菌黑色粒细胞的年龄特异性噬细胞能力

果蝇黑色素的循环训练

用于定量体外成体 果蝇 结构的快速、简单的工作流程

用于定量体外成体 果蝇 结构的快速、简单的工作流程

与神经退化有关的成人果蝇的可视化突触变性

用于定量体外成体果蝇结构的快速、简单的工作流程

用于定量体外成体果蝇结构的快速、简单的工作流程