Fast and reliable mini-prep RNA extraction from Neurospora crassa
Fast and reliable mini-prep RNA extraction from Neurospora crassa
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从粗糙脉孢菌中快速、可靠地小量制备 RNA 提取
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发表时间：
1990
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通讯作者：
V. Russo
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作者：
V. Sokolovsky;R. Kaldenhoff;M. Ricci;V. Russo
我们开发了一种从粗糙脉孢菌菌丝体中分离出高质量总 RNA 的方法，该方法能够可靠地产生大量总 RNA。一次可以提取 50 多个小量样品。知识共享许可 本作品根据知识共享署名-相同方式共享 4.0 许可获得许可。这篇常规论文可在真菌遗传学报告中找到：http://newprairiepress.org/fgr/vol37/iss1/27 从粗糙脉孢菌中快速可靠地小量制备 RNA 提取 从粗糙脉孢菌中快速可靠地小量制备 RNA V. Sokolovsky+、R. Kaldenhoff、M. Ricci 和 V.E.A.鲁索·马克斯·普朗克分子遗传学研究所 + 离开 A.N.生物化学研究所，33 Leninsky Prospekt，莫斯科 117071，苏联 我们开发了一种从粗糙脉孢菌菌丝体中分离高质量总 RNA 的方法，该方法能够可靠地产生大量的总 RNA。一次可以提取 50 多个小量样品。迄今为止发表的 RNA 提取程序大致遵循三种不同的方法，其中使用苯酚/氯仿、胍盐和/或 LiCl。然而，这些协议有一些缺点。它们要么耗时，要么产生少量 RNA，或者对于含有高水平核酸酶的培养物来说不够强大。用LiCl方法(Chambers和Russo，Fungal Genet.Newsl.1987.33：22-24)从饥饿培养物或平板菌丝体中提取，通过凝胶电泳判断得到完全降解的RNA。为了改进该方法，我们修改了 Gromoff 等人用于衣藻的实验方案。 (Mol. Cell. Biol. 1989. 9:3911-3918) 结合了苯酚萃取和 LiCl 沉淀的优点。该程序适用于快速生长的菌丝体、饥饿菌丝体、来自平板的菌丝体（4 天龄）以及在山梨糖平板上生长的粗糙脉孢菌单菌落。通过该方案，在液体培养基或平板上生长的 20 至 300 mg（干重）菌丝体可产生 200 μg 至 3 mg 的 RNA，具体取决于生长条件和菌株。我们可以从单个菌落中分离出高达 5 μg 的总 RNA。从液体或表面生长的培养物中收获菌丝体，洗涤，用滤纸彻底干燥并冷冻在液氮中。使用倒置巴斯德吸管除去包括琼脂的单菌落并冷冻。下面给出的提取程序是针对 Eppendorf 管描述的，但也可以扩大体积。如果没有另外说明，所有操作均在室温下进行。用液氮在研钵中粉碎菌丝体，将粉末转移到 2 ml Eppendorf 管中的 0.75 ml 裂解缓冲液（0.6 M NaCl、10 mM EDTA、100 mM Tris HCl，pH 8.0、4% SDS）和 0.75 ml 苯酚（用 0.1 M Tris HCl，pH 8.0 饱和）的混合物中。管内可装满菌丝体粉末 摇动 15-20 分钟 (Eppendorf Rotationsmischer 3300) 离心 10 分钟，10,000 rpm 将上相转移至等体积苯酚（用 0.1 M Tris HCl 饱和，pH 8.0）中并涡旋 离心 10 分钟，10,000 rpm 添加 0.75 体积8 M LiCl 加入上层相 4°C 下保存过夜 短暂涡旋并以 10,000 rpm 离心 10 分钟 将不总是可见的沉淀重悬于 0.3 ml 双蒸水中，与 0.03 ml 3 M 乙酸钠 (pH 5.2) 和 0.75 ml 乙醇混合 -20°C 下保存 2 小时或 -70°C 下 30 分钟 离心10 分钟，10,000 rpm 弃去上清液并用 70% 乙醇洗涤沉淀物 干燥 RNA 沉淀并将其重新溶解在 DEPC 处理的（二乙基聚碳酸酯）水中 将 RNA 溶液储存在 -70°C 通过分光光度测量来测定 RNA 制剂的纯度。 A260/A230和A260/A280比率为2或更大，表明不存在任何蛋白质或多糖污染。通常，在溴化乙锭染色的甲醛凝胶的槽中可以看到 RNA 制剂中存在的 DNA 痕迹。根据这一标准，通过所述方法制备的 RNA 样品不含 DNA 污染，而在我们进行的所有 200 次提取中都看到了清晰的未降解核糖体条带（见图 1）。通过与多个 32P 标记探针杂交进行的 Northern 印迹分析（图 2）和体外翻译实验（图 3）显示出清晰的信号，表明完整 mRNA 的存在。 New Prairie Press 出版，2017 年 从粗糙脉孢菌中快速可靠地小量制备 RNA 提取 图 1. 来自 10 种不同制剂（泳道 1-10）的总 RNA（每泳道 10 μg）在含有 2% 甲醛的 1.2% 琼脂糖凝胶上进行电泳。通过用溴化乙锭染色并用紫外线照射来进行可视化。主要条带对应于 28S 和 18S rRNA，微弱条带对应于 23S、16S 和 5S rRNA。图2.Northern印迹和与任意选择的克隆N6的32P标记的cDNA插入物杂交后的放射自显影图(T.Sommer等人1989.Nucl.Acids Research 17：5713-5723)。 Northern 分析是根据 R.A. 进行的。 Kroczek 和 E. Siebert 1990。分析。生物化学。 184：90-95。 http://newprairiepress.org/fgr/vol37/iss1/27 DOI: 10.4148/1941-4765.1492 从粗糙脉孢菌中快速可靠地小量制备 RNA 提取] 图 3. 35S-蛋氨酸标记的体外翻译产物的 SDS-PAGE (12.5%)。泳道 1：14C 标记的标记蛋白，分子宽度显示在左侧 (Mr x 10(-3)。泳道 2 和 3：分别从富含或贫铵培养基中生长的菌丝体制备的 RNA。RNA 探针对应于图 1 和 2 中的泳道 1 和 2。泳道 4：无 RNA 的对照翻译。致谢：我们感谢 Beate Otto 的体外翻译实验和 Niketan Pandit批判性地阅读了新草原出版社 2017 年出版的手稿。
We have developed a method for isolating high quality total RNA from N. crassa mycelia that reliably yields large quantities. It is possible to extract more than 50 minipreps at once. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol37/iss1/27 Fast and reliable mini-prep RNA extraction from Neurospora crassa Fast and reliable mini-prep RNA extraction from Neurospora crassa V. Sokolovsky+, R. Kaldenhoff, M. Ricci and V.E.A. Russo Max Planck Institut für molekulare Genetik + On leave from A.N. Institute of Biochemistry, 33 Leninsky Prospekt, Moscow 117071, USSR We have developed a method for isolating high quality total RNA from N. crassa mycelia that reliably yields large quantities. It is possible to extract more than 50 minipreps at once. Procedures of RNA extraction published so far follow roughly three different approaches, where phenol/chloroform, guanidinium salts, and/or LiCl is used. However, these protocols have some disadvantages. They are either time consuming, or yield low amounts of RNA, or are not powerful enough for cultures containing high levels of nucleases. Extraction from starved cultures or plate mycelia with the LiCl method (Chambers and Russo, Fungal Genet. Newsl. 1987. 33:22-24) gave completely degraded RNA as judged by gel electrophoresis. In trying to improve the method we modified the protocol used for Chlamydomonas by Gromoff et al. (Mol. Cell. Biol. 1989. 9:3911-3918) who combined the advantages of phenol extraction and LiCl precipitation. The procedure is suitable for rapidly grown mycelia, starved mycelia, mycelia from plates (4 days old) and single colonies of N. crassa grown on a sorbose plate. With this protocol, from 20 to 300 mg (dry weight) of mycelium, grown in liquid media or on plates, yielded between 200 μg and 3 mg of RNA depending on the growth conditions and strains. From a single colony we could isolate up to 5 μg total RNA. Mycelium from a liquid or surface grown culture was harvested, washed, dried thoroughly with filter paper and frozen in liquid nitrogen. Single colonies including agar were stamped out using an inverted Pasteur pipette and frozen. The procedure of extraction given below is described for Eppendorf tubes but it is also possible to scale up the volumes. All manipulations were performed at room temperature if not stated otherwise. Pulverize the mycelium in a mortar with liquid nitrogen Transfer the powder into a mixture of 0.75 ml lysis buffer (0.6 M NaCl, 10 mM EDTA, 100 mM Tris HCl, pH 8.0, 4% SDS) and 0.75 ml phenol (saturated with 0.1 M Tris HCl, pH 8.0) in a 2 ml Eppendorf tube. The tube can be filled with powdered mycelium Shake for 15-20 min (Eppendorf Rotationsmischer 3300) Centrifuge for 10 min, 10,000 rpm Transfer the upper phase into an equal volume of phenol (saturated with 0.1 M Tris HCl, pH 8.0) and vortex Centrifuge for 10 min, 10,000 rpm Add 0.75 volumes of 8 M LiCl to the upper phase Store overnight at 4°C Vortex briefly and centrifuge for 10 min, 10,000 rpm Resuspend the pellet, which is not always visible, in 0.3 ml double distilled water, mix with 0.03 ml 3 M Na-acetate (pH 5.2) and 0.75 ml ethanol Store at -20°C for 2 h or at -70°C for 30 min Centrifuge for 10 min, 10,000 rpm Discard the supernatant and wash the precipitate with 70% ethanol Dry the RNA pellet and redissolve it in DEPC treated (diethyl polycarbonate) water Store the RNA solution at -70°C Purity of the RNA preparations was assayed by spectrophotometric measurements. The A260/A230 and A260/A280 ratios were 2 or more, indicating the absence of any protein or polysaccharide contamination. Usually, traces of DNA present in RNA preparations can be seen in the slots of ethidium bromide stained formaldehyde gels. Based on this criterion, RNA samples prepared by the described method were free from contaminating DNA, while clear nondegraded ribosomal bands were seen in all 200 extractions we made (see Figure 1). Northern blot analysis by hybridization with several 32P-labelled probes (Figure 2) and in vitro translation experiments (Figure 3) have shown clear signals indicating the presence of intact mRNA. Published by New Prairie Press, 2017 Fast and reliable mini-prep RNA extraction from Neurospora crassa Figure 1. Total RNAs (10 μg per lane) from 10 different preparations (lanes 1-10) were electrophoresed on a 1.2% agarose gel containing 2% formaldehyde. Visualization was done by staining with ethidium bromide and irradiation with UV. The major bands correspond to 28S and 18S rRNA, faint bands to 23S, 16S and 5S rRNA. Figure 2. Autoradiogram after Northern blotting and hybridization to 32P-labelled cDNA insert of arbitrarily chosen clone N6 (T. Sommer et al. 1989. Nucl. Acids Research 17:5713-5723). Northern analysis was done according to R.A. Kroczek and E. Siebert 1990. Analyt. Biochem. 184:90-95. http://newprairiepress.org/fgr/vol37/iss1/27 DOI: 10.4148/1941-4765.1492 Fast and reliable mini-prep RNA extraction from Neurospora crassa ] Figure 3. SDS-PAGE (12.5%) of 35S-methionine-labelled in vitro translation products. Lane 1: 14C-labelled marker proteins, molecular wieghts are indicated on the left (Mr x 10(-3). Lanes 2 and 3: RNA prepared from mycelia grown in rich or ammonium depleted medium respectively. RNA probes correspond to lanes 1 and 2 in Fig. 1 and 2. Lane 4: control translation without RNA. Acknowledgements: We thank Beate Otto for in vitro translation experiments and Niketan Pandit for critically reading the manuscript. Published by New Prairie Press, 2017