EASYspin Plus 多糖多酚復(fù)雜植物RNA快速提取試劑盒操作方法及步驟說明書

1、.EASYspin Plus Complex Plant RNA KitEASYspin Plus多糖多酚/復(fù)雜植物RNA快速提取試劑盒目錄號：RN53v 適用范圍：適用于快速提取植物組織細(xì)胞總RNA，使用獨(dú)有基因組DNA清除柱技術(shù)可有效清除電泳可見gDNA殘留，RNA可用于反轉(zhuǎn)錄PCR，熒光定量PCR等。v 試劑盒組成、儲存、穩(wěn)定性：試劑盒組成保存50次(RN5301)裂解液CLB 室溫50 ml裂解液RLT Plus室溫25 ml去蛋白液RW1室溫40 ml漂洗液RW室溫10 ml第一次使用前按說明加指定量乙醇RNase-free H2O室溫10 ml基因組DNA清除柱和收集管室溫50套R

2、Nase-free吸附柱RA和收集管室溫50套本試劑盒在室溫儲存12個月不影響使用效果。儲存事項(xiàng)：1. 不合適的儲存于低溫（4或者20）會造成溶液沉淀，影響使用效果，因此運(yùn)輸和儲存均在室溫下（1525）進(jìn)行。2. 避免試劑長時間暴露于空氣中產(chǎn)生揮發(fā)、氧化、PH值變化，各溶液使用后應(yīng)及時蓋緊蓋子。注意事項(xiàng)1. 所有的離心步驟均可在室溫完成（4離心也可以），使用轉(zhuǎn)速可以達(dá)到13,000 rpm的傳統(tǒng)臺式離心機(jī)，如Eppendorf 5415C 或者類似離心機(jī)。2. 需要自備-巰基乙醇，乙醇，研缽(可選)。3. 樣品處理量絕對不要超過基因組清除柱DA和和RNA吸附柱RA處理能力，否則造成DNA殘留或

3、產(chǎn)量降低。開始摸索實(shí)驗(yàn)條件時，如果不清楚樣品DNA/RNA含量時可使用較少的樣品處理量，將來根據(jù)樣品試驗(yàn)情況增加或者減少處理量。4. 裂解液CLB和RLT Plus 和去蛋白液RW1中含有刺激性化合物，操作時戴乳膠手套，避免沾染皮膚，眼睛和衣服。若沾染皮膚、眼睛時，要用大量清水或者生理鹽水沖洗。5. 關(guān)于DNA 的微量殘留:一般說來任何總RNA提取試劑在提取過程中無法完全避免DNA的微量殘留（DNase消化也無法做到100%無殘留），本公司的EASYspin Plus RNA提取產(chǎn)品，由于采取了本公司獨(dú)特的緩沖體系和基因組DNA清除柱技術(shù)，絕大多數(shù)DNA已經(jīng)被清除，不需要DNase消化，可直接

4、用于反轉(zhuǎn)錄PCR和熒光定量PCR。個別特殊情況如DNA含量過于豐富造成殘留或者要進(jìn)行嚴(yán)格的mRNA表達(dá)量分析熒光定量PCR，我們建議在進(jìn)行模板和引物的選擇時：1) 選用跨內(nèi)含子的引物，以穿過mRNA中的連接區(qū)，這樣DNA就不能作為模板參與擴(kuò)增反應(yīng)。2) 選擇基因組DNA和cDNA上擴(kuò)增的產(chǎn)物大小不一樣的引物對。3) 將RNA提取物用RNase-free的DNase I 處理。本試劑盒還可以用于DNase I處理后的RNA清潔(cleanup) ，請聯(lián)系我們索取具體操作說明書。4) 在步驟去蛋白液RW1漂洗前，直接在吸附柱RA上進(jìn)行DNase I柱上消化處理。購買DNA酶柱上消化試劑盒(貨號：R

5、N34)前可先索取具體操作說明書。v 操作步驟：（實(shí)驗(yàn)前請先閱讀注意事項(xiàng)）提示：ð 第一次使用前請先在漂洗液RW瓶加入指定量無水乙醇!ð 取1ml裂解液 CLB至離心管內(nèi)(如果CLB有析出或者沉淀需先置于65°C水浴重新溶解），在裂解液CLB中加入5% -巰基乙醇（1ml CLB加50l -巰基乙醇）。顛倒混勻后65°C水浴中預(yù)熱。多個樣品按照比例放大準(zhǔn)備。1. 直接研磨法（實(shí)驗(yàn)室無液氮情況下或者柔軟易研磨植物樣品推薦此法）:a. 新鮮植物組織或者冰凍保存樣品稱重后取100-200mg（水分少的樣品如葉片種子等可加100-150mg，水分多的樣品如草莓西

6、瓜果實(shí)可多加一些）迅速剪成小塊放入研缽，加入 1ml CLB（已加有-巰基乙醇）室溫下充分研磨成勻漿，注意應(yīng)該迅速研磨讓組織和裂解液CLB立刻充分接觸以抑制RNA酶活性。-巰基乙醇是裂解液CLB的關(guān)鍵成分，必要的時候可以提高終濃度到10-20%。如果特別復(fù)雜植物，可以嘗試在裂解液中加入PVP40至終濃度2%。b. 將裂解物轉(zhuǎn)入離心管，立即劇烈振蕩15秒，短時放回 65°C水浴中（5-10 min），中間偶爾顛倒1-2次幫助裂解。13，000rpm離心10分鐘，沉淀不能裂解的碎片。c. 取裂解物上清（在不超過基因組DNA清除柱能力的情況下可以取更多的上清，這樣可以提高產(chǎn)量）轉(zhuǎn)到一個新離

7、心管。加入上清體積一半的無水乙醇(0.5體積)，此時可能出現(xiàn)沉淀，但是不影響提取過程，立即吹打混勻，不要離心。若上清表面有漂浮物，用吸頭挑開吸取下面液體即可。d. 立刻接操作步驟的步驟3。2. 液氮研磨法（適用廣泛，提取復(fù)雜難破碎，易降解樣品時推薦此法）:a. 液氮中研磨新鮮或-70°C冷凍的材料至細(xì)粉。b. 轉(zhuǎn)移100-200mg細(xì)粉（水分少的樣品如葉片種子等可加100-150mg，水分多的樣品如草莓西瓜果實(shí)可多加一些）加至預(yù)熱的裂解液CLB（已加有-巰基乙醇）離心管中。立即劇烈渦旋30-60秒或者用吸頭吹打混勻裂解直得到滿意勻漿結(jié)果(或者電動勻漿30秒)，可以剪切DNA，降低粘稠

8、度和提高產(chǎn)量。c. 短時放回 65°C水浴中（5-10 min），中間偶爾顛倒1-2次幫助裂解。d. 將裂解物13,000 rpm離心10分鐘，沉淀不能裂解的碎片。e. 取裂解物上清（在不超過基因組DNA清除柱能力的情況下可以取更多的上清，這樣可以提高產(chǎn)量）轉(zhuǎn)到一個新離心管。加入上清體積一半的無水乙醇(0.5體積)，此時可能出現(xiàn)沉淀，但是不影響提取過程，立即吹打混勻，不要離心。若上清表面有漂浮物，用吸頭挑開吸取下面液體即可。f. 立刻接操作步驟的步驟3。3. 將混合物(每次小于720l，多可以分兩次加入)加入一個基因組清除柱中，（清除柱放入收集管中）13,000

9、rpm離心2分鐘，棄掉廢液。確保離心后液體全部濾過去，膜上沒有殘留，如有必要，可以加大離心力和離心時間。4. 將基因組DNA清除柱子放在一個干凈2ml離心管內(nèi)（不用RNAse free 或者DEPC處理，一般干凈的新離心管即可。也可使用RNA吸附柱配套的新的干凈收集管），在基因組清除柱內(nèi)加500l裂解液RLT Plus，13,000 rpm離心30秒， 收集濾液（RNA在濾液中），用微量移液器較精確估計(jì)濾過液體積（通常為450-500l左右，濾過時候損失體積應(yīng)該減去），加入0.5倍體積的無水乙醇，此時可能出現(xiàn)沉淀，但是不影響提取過程，立即吹打混勻，不要離心。5. 立刻將混合物(每次小于720l

10、，多可以分兩次加入)加入一個吸附柱RA中，（吸附柱放入收集管中）13,000 rpm離心2分鐘，棄掉廢液。確保離心后液體全部濾過去，膜上沒有殘留，如有必要，可以加大離心力和離心時間。6. 加700l 去蛋白液RW1，室溫放置1分鐘，13,000rpm 離心30秒，棄掉廢液。7. 加入500l漂洗液RW（請先檢查是否已加入無水乙醇!），13,000 rpm 離心30秒，棄掉廢液。加入500l漂洗液RW，重復(fù)一遍。8. 將吸附柱RA放回空收集管中，13,000 rpm離心2分鐘，盡量除去漂洗液, 以免漂洗液中殘留乙醇抑制下游反應(yīng)。9. 取出吸附柱RA，放入一個RNase free離心管中，根據(jù)預(yù)期

11、RNA產(chǎn)量在吸附膜的中間部位加30-50l RNase free water（事先在 70-90水浴中加熱可提高產(chǎn)量）， 室溫放置1分鐘，12,000 rpm 離心1分鐘。10. 如果預(yù)期RNA產(chǎn)量>30g，加30-50l RNase free water重復(fù)步驟9，合并兩次洗液，或者使用第一次的洗脫液加回到吸附柱重復(fù)步驟一遍(如果需要RNA濃度高)。洗脫兩遍的RNA洗脫液濃度高,分兩次洗脫合并洗脫液的RNA產(chǎn)量比前者高1530%,但是濃度要低,用戶根據(jù)需要選擇。附錄1：DNA酶柱上消化（詳細(xì)請參考RN34 DNase I 柱上消化試劑盒說明書)1. 按照前面所列RN53試劑盒操作步驟操

12、作，直到做完操作步驟5。2. 取45l DNase I buffer和5l RNase free DNase I在離心管輕輕吹打混勻成工作液（處理多個離心柱子要按照比例放大制備工作液）。3. 向吸附柱RA 中加入350l去蛋白液RW1，12,000 rpm 離心30 秒，棄廢液，將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，室溫（20-30）放置15 分鐘。注意直接將工作液滴在膜中央上向膜四周浸潤充分和膜接觸，不要讓工作液滴在O型墊圈或是離心柱管壁上掛壁或者掛在墊圈上不能充分和膜接觸。5. 向吸附柱RA 中加入350l去蛋白液RW1， 12,000 rpm

13、離心30-60 秒，棄廢液，將吸附柱放回收集管中。6. 接操作步驟7完成后續(xù)步驟。附錄2：RNA含量少樣品或者RNA復(fù)雜產(chǎn)量低的解決方案 可以提高樣品處理量到300-500mg/2ml裂解液CLB，上清過兩根基因組DNA清除柱子，洗脫下來的RNA，可以兩個合并到一根RNA吸附柱上，可以大大提高RNA濃度。附錄3：使用EASYspin/EASYspin Plus植物RNA提取系列試劑盒發(fā)表文章100多篇：1. 桃果實(shí)、花、根、葉：Isolation, characterisation and phylogenetic analysis of resistance gene  

14、  analogues in a wild species of peach (Prunus kansuensis).Canadian Journal of Plant Science,    2011, 91(6): 961-970 2. 櫻桃花、葉、顎等各部位：Over-expression of the PaAP1 gene from sweet cherry (Prunus avium    L.)    causes early floweri.Journal of Plant Phy

15、siology, 2012,Available online 1 December    2012 3. 洋蔥根、莖、蕾、葉、雌雄蕊等各部位：Cloning and Expression Analysis of A Putative B Class MADS-box    Gene of AcPI in Onion. Scientia Agricultura Sinica, 2012, 45(23):4759-4769 4. 蕪菁：Isolation and Functional Characterisation of the Gen

16、es Encoding 8-Sphingolipid       Desaturase from Brassica rapa. Journal of Genetics and Genomics Volume 39, Issue 1, January    2012,    Pages 4759 5. 蕪 菁 1 ：EXPRESSION, DIVERGENCE AND EVOLUTION OF THE CALEOSIN GENE FAMILY IN BRASSICA RAPA.

17、 Arch. Biol. Sci., Belgrade, 65 (3), 863-876, 2013 DOI:10.2298/ABS1303863H6. 番茄葉：Effect of Low Temperature Stress on the Expression of ProDH Gene and the Activities of the Proline Dehydrogenase in Leaves of Tomato Seedling. Chinese Agricultural Science  Bulletin 2012,28(10):132-135 7. 梔子葉：

18、Isolation of High Quality Total RNA from Gardenia jasminoides Eills.Chinese Agricultural    Science Bulletin.2012, 28(27):194-198 8. 油桐果實(shí)：Cui Qinqin, Han Xiaojiao, Chen Yicun, Zhan Zhiyong, Lin Liyuan, Wang Yangdong.     Isolation and Expression Characteristics of

19、Biotin Carboxyl Carrier Protein Coding Gene（VfBCCP)  from Vernicia fordii.SCIENTIA SILVAE SINICAE. 2012, 48(8): Available online August 9. 油桐果實(shí)1：Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time    PCR in Tung Tree during Seed Development. PLoS ONE

20、, 2012, 7(8): e43084 10. 紫菜：Molecular cloning and expression analysis of ribosomal protein S7 gene from Porphyra    haitanensis. JOURNAL OF FISHERIES OF CHINA, 2011, 35（12）：1814-1821 11. 石斛：Molecular characterization of a mitogen-activated protein kinase gene DoMPK1 in Dendrobium offi

21、cinale. Acta Pharmaceutica Sinica, 2012, 47 (12): 1703-1709 12. 石斛1：ESTs Analysis Reveals Putative Genes Involved in Symbiotic Seed Germination in Dendrobium officinale. Symbiotic Germination Genes in D. officinale. August 2013 | Volume 8 | Issue 8 | e7270513. 大豆：RNA-seq Analysis Reveals Ethylene-Me

22、diated Reproductive Organ Development and Abscission in Soybean(Glycine max L. Merr.). Plant Mol Biol Rep, 2012, published online: 4 Dec, 2012 14. 大豆1：Construction of ethylene regulatory network based on the phytohormones related gene transcriptome profiling and prediction of transcription factor ac

23、tivities in soybean. Acta Physiol Plant, 2012, published online: 12 Dec, 2012 15. 紅花玉蘭：Expression Analysis of MAwuAG in Different Organs and Developmental Stages of Magnolia wufengensis. Chinese Bulletin of Botany, 2013, 48 (2): 15 16. 毛桃：Cloning and Phylogeny Analysis of PpAP2 Floral Homologous Gen

24、es in Peach. Chinese Agricultural Science Bulletin, 2013, 29(7): 99-104 17. 五倍子：Cloning and characterisation of a phenylalanine ammonia-lyase gene from Rhus chinensis. Plant Cell Rep, 2013, published online：15 March, 2013 18. :五倍子1：Cloning, characterization and expression of chalcone synthase from m

25、edicinal plant Rhus chinensis.J. Plant Biochem. Biotechnol. DOI 10.1007/s13562-013-0231-919. 青杄 ：cDNA Cloning and Bioinformatic Analysis of the sPPa1 Gene form Picea wilsonii. Plant Science Journal, 2012, 30(40): 394-401 20. 青杄 1：cDNA Cloning and Bioinformatic Analysis of PsbO Gene from Picea wilson

26、ii.Life Science Research, 2012, 16(3): 201-20621. 青杄 2：Cloning and Tissue Expression Analysis of PwPSAF in Picea wilsonii. SCIENTIA SILVAE SINICAE. Vol. 49，No. 10, Oct. 2013.22. 洋蔥：Molecular Cloning and Transcriptional Analysis of the Putative AGAMOUS Homolog AcAG in Onion (Allium cepa. Plant Mol Bi

27、ol Rep, DOI 10.1007/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 responsibl

28、e for its seed 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 Promoter

29、s in Transgenic 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/s11240-013-04

30、23-y.28. 茶梅花瓣：Comparison and Analysis of Methods of Extracting Total RNA from Petals of Camellia sasanqua. Chinese Agricultural Science Bulletin.2013,29(28):129-133.29. 梔子：Isolation of High Quality Total RNA fromGardenia jasminoides Eills. Chinese Agricultural Science Bulletin. 2012, 28(27):194-1983

31、0. 丹參：Genome-wide analysis and molecular dissection of the SPL gene family in Salvia miltiorrhiza. 2014 Jan;56(1):38-50. doi: 10.1111/jipb.12111. Epub 2013 Nov 20.31. 牡丹：Transcriptome Comparison Reveals Key Candidate Genes Responsible for the Unusual Reblooming Trait in Tree Peonies. Genes Responsib

32、le for Reblooming in Tree Peonies. November 2013 | Volume 8 | Issue 11 | e7999632. 東南景天：Role of sulfur assimilation pathway in cadmium hyperaccumulation by Sedum alfredii Hance. Ecotoxicology and Environmental Safety. Volume 100, February 2014, Pages 159165.33. 山蒼子：Identification of appropriate refe

33、rence genes for 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

34、 studies in woody 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

35、 and defence responses during the development of peach gummosis caused by Lasiodiplodia theobromae. Eur J Plant Pathol (2014) 138:195207, DOI 10.1007/s10658-013-0322-4.37. 桃子1：Carbohydrate metabolism changes in Prunus persica gummosis infected with Lasiodiplodia theobromae. Phytopathology "Firs

36、t Look" paper /10.1094/PHYTO-01-13-0025-R posted 11/27/2013.38. 海棠：The Malus crabapple transcription factor McMYB10 regulatesanthocyanin biosynthesis during petal coloration. Scientia Horticulturae 166 (2014) 4249.39. 海藻：A rapid and sensitive method for field detection of Proroc

37、entrum donghaiense using reverse transcription-coupled loop-mediated isothermal amplification. Harmful Algae 29 (2013) 3139.40. 油茶：Establish a cDNA-AFLP Technology System in Camellia oleifera. Molecular Plant Breeding, 2013, Vol.11, No.5, 611-616.41. 亞洲百合：Transcriptomic analysis of Asiatic lily in t

38、he process of vernalization 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. 人參：Cloning and Sequence Analysis Squalene Epoxidase Gene

39、in Panax gin-seng. Journal of Jilin Agricultural University 2014, 36(2): 149-152,1744. 雪蓮：Cloning and Sequence Analysis of rbcs Gene from Sasussured involucrdta Kar. et Kir. Chinese Agricultural Science Bulletin 2014, 30(15): 261-26745. 柑橘3：Secreted Expression of Cecropin B Gene Enhances Resistance

40、to Xanthomonas axonopodis pv. citri in Transgenic Citrus sinensisTarocco Acta Horticulturae Sinica 2014, 41(3): 417428 http: / www. ahs. ac. cn46. 菊花：Stem apex detoxification culture markedly improved severalphysiological characters of chrysanthemum YUTAI. Plant Cell Tiss Organ Cult 2014, DOI 10.100

41、7/s11240-014-0541-147. 蕎麥和擬南芥：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 fe

42、rtile clone of Pinus tabulaeformis. 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. 棉花和擬南芥：Functional characterizatio

43、n of GhAKT1, a novel Shaker-like K+ channel gene involved in K+ uptake from cotton (Gossypium hirsutum). Gene 2014, 545(1): 617151. 棉花和擬南芥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. 白楊