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1、杭州昊鑫生物科技股份有限公司 htpp:/EASYspin Plus Complex Plant RNA KitEASYspin Plus多糖多酚/复杂植物RNA快速提取试剂盒目录号：RN53v 适用范围：适用于快速提取植物组织细胞总RNA，使用独有基因组DNA清除柱技术可有效清除电泳可见gDNA残留，RNA可用于反转录PCR，荧光定量PCR等。v 试剂盒组成、储存、稳定性：试剂盒组成保存50次(RN5301)裂解液CLB 室温50 ml裂解液RLT Plus室温25 ml去蛋白液RW1室温40 ml漂洗液RW室温10 ml第一次使用前按说明加指定量乙醇RNase-free H2O室温10 m
2、l基因组DNA清除柱和收集管室温50套RNase-free吸附柱RA和收集管室温50套本试剂盒在室温储存12个月不影响使用效果。储存事项：1. 不合适的储存于低温（4或者20）会造成溶液沉淀，影响使用效果，因此运输和储存均在室温下（1525）进行。2. 避免试剂长时间暴露于空气中产生挥发、氧化、PH值变化，各溶液使用后应及时盖紧盖子。注意事项1. 所有的离心步骤均可在室温完成（4离心也可以），使用转速可以达到13,000 rpm的传统台式离心机，如Eppendorf 5415C 或者类似离心机。2. 需要自备-巯基乙醇，乙醇，研钵(可选)。3. 样品处理量绝对不要超过基因组清除柱DA和和RNA
3、吸附柱RA处理能力，否则造成DNA残留或产量降低。开始摸索实验条件时，如果不清楚样品DNA/RNA含量时可使用较少的样品处理量，将来根据样品试验情况增加或者减少处理量。4. 裂解液CLB和RLT Plus 和去蛋白液RW1中含有刺激性化合物，操作时戴乳胶手套，避免沾染皮肤，眼睛和衣服。若沾染皮肤、眼睛时，要用大量清水或者生理盐水冲洗。5. 关于DNA 的微量残留:一般说来任何总RNA提取试剂在提取过程中无法完全避免DNA的微量残留（DNase消化也无法做到100%无残留），本公司的EASYspin Plus RNA提取产品，由于采取了本公司独特的缓冲体系和基因组DNA清除柱技术，绝大多数DNA
4、已经被清除，不需要DNase消化，可直接用于反转录PCR和荧光定量PCR。个别特殊情况如DNA含量过于丰富造成残留或者要进行严格的mRNA表达量分析荧光定量PCR，我们建议在进行模板和引物的选择时：1) 选用跨内含子的引物，以穿过mRNA中的连接区，这样DNA就不能作为模板参与扩增反应。2) 选择基因组DNA和cDNA上扩增的产物大小不一样的引物对。3) 将RNA提取物用RNase-free的DNase I 处理。本试剂盒还可以用于DNase I处理后的RNA清洁(cleanup) ，请联系我们索取具体操作说明书。4) 在步骤去蛋白液RW1漂洗前，直接在吸附柱RA上进行DNase I柱上消化处
5、理。购买DNA酶柱上消化试剂盒(货号：RN34)前可先索取具体操作说明书。v 操作步骤：（实验前请先阅读注意事项）提示： 第一次使用前请先在漂洗液RW瓶加入指定量无水乙醇! 取1ml裂解液 CLB至离心管内(如果CLB有析出或者沉淀需先置于65C水浴重新溶解），在裂解液CLB中加入5% -巯基乙醇（1ml CLB加50l -巯基乙醇）。颠倒混匀后65C水浴中预热。多个样品按照比例放大准备。1. 直接研磨法（实验室无液氮情况下或者柔软易研磨植物样品推荐此法）:a. 新鲜植物组织或者冰冻保存样品称重后取100-200mg（水分少的样品如叶片种子等可加100-150mg，水分多的样品如草莓西瓜果实可
6、多加一些）迅速剪成小块放入研钵，加入 1ml CLB（已加有-巯基乙醇）室温下充分研磨成匀浆，注意应该迅速研磨让组织和裂解液CLB立刻充分接触以抑制RNA酶活性。-巯基乙醇是裂解液CLB的关键成分，必要的时候可以提高终浓度到10-20%。如果特别复杂植物，可以尝试在裂解液中加入PVP40至终浓度2%。b. 将裂解物转入离心管，立即剧烈振荡15秒，短时放回 65C水浴中（5-10 min），中间偶尔颠倒1-2次帮助裂解。13，000rpm离心10分钟，沉淀不能裂解的碎片。c. 取裂解物上清（在不超过基因组DNA清除柱能力的情况下可以取更多的上清，这样可以提高产量）转到一个新离心管。加入上清体积一
7、半的无水乙醇(0.5体积)，此时可能出现沉淀，但是不影响提取过程，立即吹打混匀，不要离心。若上清表面有漂浮物，用吸头挑开吸取下面液体即可。d. 立刻接操作步骤的步骤3。2. 液氮研磨法（适用广泛，提取复杂难破碎，易降解样品时推荐此法）:a. 液氮中研磨新鲜或-70C冷冻的材料至细粉。b. 转移100-200mg细粉（水分少的样品如叶片种子等可加100-150mg，水分多的样品如草莓西瓜果实可多加一些）加至预热的裂解液CLB（已加有-巯基乙醇）离心管中。立即剧烈涡旋30-60秒或者用吸头吹打混匀裂解直得到满意匀浆结果(或者电动匀浆30秒)，可以剪切DNA，降低粘稠度和提高产量。c. 短时放回65
8、C水浴中（5-10min），中间偶尔颠倒1-2次帮助裂解。d. 将裂解物13,000 rpm离心10分钟，沉淀不能裂解的碎片。e. 取裂解物上清（在不超过基因组DNA清除柱能力的情况下可以取更多的上清，这样可以提高产量）转到一个新离心管。加入上清体积一半的无水乙醇(0.5体积)，此时可能出现沉淀，但是不影响提取过程，立即吹打混匀，不要离心。若上清表面有漂浮物，用吸头挑开吸取下面液体即可。f. 立刻接操作步骤的步骤3。3. 将混合物(每次小于720l，多可以分两次加入)加入一个基因组清除柱中，（清除柱放入收集管中）13,000 rpm离心2分钟，弃掉废液。确保离心后液体全部滤过去，膜上没有残留，
9、如有必要，可以加大离心力和离心时间。4. 将基因组DNA清除柱子放在一个干净2ml离心管内（不用RNAse free 或者DEPC处理，一般干净的新离心管即可。也可使用RNA吸附柱配套的新的干净收集管），在基因组清除柱内加500l裂解液RLT Plus，13,000 rpm离心30秒， 收集滤液（RNA在滤液中），用微量移液器较精确估计滤过液体积（通常为450-500l左右，滤过时候损失体积应该减去），加入0.5倍体积的无水乙醇，此时可能出现沉淀，但是不影响提取过程，立即吹打混匀，不要离心。5. 立刻将混合物(每次小于720l，多可以分两次加入)加入一个吸附柱RA中，（吸附柱放入收集管中）13
10、,000 rpm离心2分钟，弃掉废液。确保离心后液体全部滤过去，膜上没有残留，如有必要，可以加大离心力和离心时间。6. 加700l 去蛋白液RW1，室温放置1分钟，13,000rpm 离心30秒，弃掉废液。7. 加入500l漂洗液RW（请先检查是否已加入无水乙醇!），13,000 rpm 离心30秒，弃掉废液。加入500l漂洗液RW，重复一遍。8. 将吸附柱RA放回空收集管中，13,000 rpm离心2分钟，尽量除去漂洗液, 以免漂洗液中残留乙醇抑制下游反应。9. 取出吸附柱RA，放入一个RNase free离心管中，根据预期RNA产量在吸附膜的中间部位加30-50l RNase free w
11、ater（事先在 70-90水浴中加热可提高产量）， 室温放置1分钟，12,000 rpm 离心1分钟。10. 如果预期RNA产量30g，加30-50l RNase free water重复步骤9，合并两次洗液，或者使用第一次的洗脱液加回到吸附柱重复步骤一遍(如果需要RNA浓度高)。洗脱两遍的RNA洗脱液浓度高,分两次洗脱合并洗脱液的RNA产量比前者高1530%,但是浓度要低,用户根据需要选择。附录1：DNA酶柱上消化（详细请参考RN34 DNase I 柱上消化试剂盒说明书)1. 按照前面所列RN53试剂盒操作步骤操作，直到做完操作步骤5。2. 取45l DNase I buffer和5l
12、RNase free DNase I在离心管轻轻吹打混匀成工作液（处理多个离心柱子要按照比例放大制备工作液）。3. 向吸附柱RA 中加入350l去蛋白液RW1，12,000 rpm 离心30 秒，弃废液，将吸附柱放回收集管中。4. 向吸附柱RA 中央加入50l的DNase I 工作液，室温（20-30）放置15 分钟。注意直接将工作液滴在膜中央上向膜四周浸润充分和膜接触，不要让工作液滴在O型垫圈或是离心柱管壁上挂壁或者挂在垫圈上不能充分和膜接触。5. 向吸附柱RA 中加入350l去蛋白液RW1， 12,000 rpm 离心30-60 秒，弃废液，将吸附柱放回收集管中。6. 接操作步骤7完成后续
13、步骤。附录2：RNA含量少样品或者RNA复杂产量低的解决方案 可以提高样品处理量到300-500mg/2ml裂解液CLB，上清过两根基因组DNA清除柱子，洗脱下来的RNA，可以两个合并到一根RNA吸附柱上，可以大大提高RNA浓度。附录3：使用EASYspin/EASYspin Plus植物RNA提取系列试剂盒发表文章100多篇：1. 桃果实、花、根、叶：Isolation, characterisation and phylogenetic analysis of resistance gene analogues in a wild species of peach (Prunus kans
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26、.1007/s11105-013-0607-y23. 木瓜：XsFAD2 gene encodes the enzyme responsible for the high linoleic acid content in oil accumulated in Xanthoceras sorbifolia seeds. JOURNAL ARTICLE. 2013-6-17.24. 木瓜1：Two novel diacylglycerol acyltransferase genes from Xanthoceras 2 sorbifolia are responsible for its seed
27、 oil content. GENE-38688; No. of pages: 9; 4C:25. 柑橘：Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection. Plant Cell Rep, DOI 10.1007/s00299-013-1470-x26. 柑橘1：Expression Analysis of Three Phloem-specific Promoters in Transgeni
28、c Poncirus trifoliata. Acta Horticulturae Sinica. 2014, 41(1): 18.27. 柑橘2： Activation of three pathogen-inducible promoters in transgenic citrus (Citrus sinensis Osbeck) after Xanthomonas axonopodis pv. citri infection and wounding. Plant Cell Tiss Organ Cult. DOI 10.1007/s11240-013-0423-y.28. 茶梅花瓣：
29、Comparison and Analysis of Methods of Extracting Total RNA from Petals of Camellia sasanqua. Chinese Agricultural Science Bulletin.2013,29(28):129-133.29. 栀子：Isolation of High Quality Total RNA fromGardenia jasminoides Eills. Chinese Agricultural Science Bulletin. 2012, 28(27):194-19830. 丹参：Genome-w
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31、ing in Tree Peonies. November 2013 | Volume 8 | Issue 11 | e7999632. 东南景天：Role of sulfur assimilation pathway in cadmium hyperaccumulation by Sedum alfredii Hance. Ecotoxicology and Environmental Safety. Volume 100, February 2014, Pages 159165.33. 山苍子：Identification of appropriate reference genes fo
32、r normalizing transcript expression by quantitative realtime PCR in Litsea cubeba. TECHNICAL NOTE. Mol Genet Genomics (2013) 288:727737, DOI 10.1007/s00438-013-0785-134. 木本植物：Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in wo
33、ody plants. ORIGINAL PAPER. Plant Cell Tiss Organ Cult, DOI 10.1007/s11240-013-0393-035. 棉花：Analysis of sea-island cotton and upland cotton in response to Verticillium dahliae infection by RNA sequencing. Sun et al. BMC Genomics 2013, 14:852 /1471-2164/14/852.36. 桃子：Biochemical changes and defence r
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37、n via RNA-seq. Mol Biol Rep. DOI 10.1007/s11033-014-3250-2.42. 毛泡桐：Dynamic expression of novel and conserved microRNAs and their targets in diploid and tetraploid of Paulownia tomentosa. Biochimie xxx (2014) 1e10.43. 人参：Cloning and Sequence Analysis Squalene Epoxidase Gene in Panax gin-seng. Journal
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40、拟南芥：Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant. Gene 2014, 550(2): 20020648. 油松：Differential expression of SLOW WALKER2 homologue in ovules of female sterile mutant and fertile clone of Pinus tabul
41、aeformis. Russian Journal of Developmental Biology 2014, 45(2): 78-84 49. 玫瑰花：Precise spatio-temporal modulation of ACC synthase by MPK6 cascade mediates the response of rose flowers to rehydration. The Plant Journal 2014, 79(6): 941950 50. 棉花和拟南芥：Functional characterization of GhAKT1, a novel Shake
42、r-like K+ channel gene involved in K+ uptake from cotton (Gossypium hirsutum). Gene 2014, 545(1): 617151. 棉花和拟南芥1：Upland Cotton Gene GhFPF1 Confers Promotion of Flowering Time and Shade-Avoidance Responses in Arabidopsis thaliana. PLoS ONE 2014, 9(3): e91869. doi:10.1371/journal.pone.009186952. 白杨：P
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46、tform. PLoS ONE 2014, 9(3): e90636. doi:10.1371/journal.pone.009063658. 棉花1：Gibberellin Overproduction Promotes Sucrose Synthase Expression and Secondary Cell Wall Deposition in Cotton Fibers. PLoS ONE 2014, 9(5): e96537. doi:10.1371/journal.pone.0096537 59. 苹果：Low Medium pH Value Enhances Anthocyanin Accumulation in Malus Crabapple Leaves. PLoS ONE 2014, 9(6): e97904. doi:10.1371/journal.pone.009790460. 毛果杨：Three homologous genes encoding functional D8-sphingolipid desaturase in Populus tomentosa. Genes Genom 2014, 36: 293301n