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RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis
单细胞绿藻佐夫色绿藻中的RNA纯化   

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参见作者原研究论文

本实验方案简略版
Proceedings of the National Academy of Sciences of the United States of America
May 2017

Abstract

Chromochloris zofingiensis is a unicellular green alga that is an emerging model species for studies in fields such as biofuel production, ketocarotenoid biosynthesis and metabolism. The recent availability of a high-quality genome assembly facilitates systems-level analysis, such as RNA-Seq. However, cells of this alga have a tough cell wall, which is a challenge for RNA purification. This protocol was designed specifically to breach the cell wall and isolate high-quality RNA suitable for RNA-Seq studies.

Keywords: Chromochloris zofingiensis (佐夫色绿藻), Algae (藻类), RNA purification (RNA纯化), RNA-Seq (RNA-Seq), Cell wall (细胞壁)

Background

Chromochloris zofingiensis is a small unicellular green alga from the chlorophyte lineage (Dönz, 1934). Previously, this species has been described in the literature with the genera Muriella, Chlorella, and Mychonastes (Fučíková and Lewis, 2012). There are strong economic interests in C. zofingiensis because it is capable of producing large quantities of lipids for biofuels and shows promise as a source of the commercially valuable nutraceutical astaxanthin (Breuer et al., 2012; Mulders et al., 2014; Liu et al., 2016). Recently, a high-quality, chromosome-level genome assembly and accompanying annotations were published, which facilitates systems level analyses like RNA-Seq (Roth et al., 2017). The following protocol was designed to produce highly purified total RNA, including miRNA, from liquid cultures of C. zofingiensis suitable for RNA-Seq. C. zofingiensis cells are protected by a robust cell wall, which this protocol was designed to penetrate. Starting material for the protocol can be up to 2.5 x 108 total cells. This protocol should yield at least 20 µg of highly purified RNA suitable for the preparation of RNA-Seq libraries by standard kits, such as the Illumina TruSeq Stranded Total RNA kit.

Materials and Reagents

  1. 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
  2. Serological pipettes, 10 ml (Fisher Scientific, catalog number: 13-676-10J )
  3. Lysing Matrix D Tubes (2 ml) (MP Biomedicals, catalog number: 116913050 )
  4. Tapered end, metal spatula (Fisher Scientific, catalog number: 14-374 )
  5. VacConnectors (QIAGEN, catalog number: 19407 )
  6. C. zofingiensis (SAG, catalog number: 211-14 )
  7. Proteose medium (UTEX Culture Collection of Algae, Proteose medium)
  8. Chu’s medium (UTEX Culture Collection of Algae, Chu’s medium)
  9. Liquid nitrogen (LN2) (various)
  10. Wet ice (various)
  11. Dry ice (various)
  12. RNaseZap (250 ml spray bottle) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 )
  13. RNase-free ethanol (EtOH) (100% and 70%) (various)
  14. TRIzol (100 ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596026 )
  15. Chloroform:isoamyl alcohol (24:1) (Sigma-Aldrich, catalog number: C0549-1PT )
  16. MaXtract High Density (100 x 15 ml) (QIAGEN, catalog number: 129065 )
  17. miRNeasy Mini Kit (50) (QIAGEN, catalog number: 217004 ), which includes:
    1. QIAGEN miRNeasy mini columnsa.QIAGEN miRNeasy mini columns
    2. 1.5 and 2 ml collection tubes
    3. QIAGEN buffer RWT
    4. QIAGEN buffer RPE
    5. RNase-free water
  18. RNase-Free DNase Set (50 samples) (QIAGEN, catalog number: 79254 ), which includes:
    1. RNase-free DNase I
    2. QIAGEN buffer RDD
  19. 3 M sodium acetate (NaOAc), pH = 8, RNase-free (various)
  20. 1 M Tris hydrochloride (Tris-HCl), pH = 8, RNase-free (various)
  21. 5 M sodium chloride (NaCl), RNase-free (various)
  22. 0.5 M ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), pH = 8, RNase-free (various)
  23. 10% sodium dodecyl sulfate (SDS) (various)
  24. 20 mg/ml Proteinase K (Fisher Scientific, catalog number: BP1700-100 )
  25. Agilent RNA 6000 Nano kit (Agilent Technologies, catalog number: 5067-1511 )
  26. Lysis buffer (see Recipes)

Equipment

  1. Refrigerated centrifuge (Eppendorf, model: 5810 R )
  2. Refrigerated microfuge (Eppendorf, model: 5424 R )
  3. Clean bench
  4. Ice buckets (Corning, catalog number: 432122 )
  5. PIPETMAN Classic pipet (Gilson, model: P20, catalog number: F123600 )
  6. PIPETMAN Classic pipet (Gilson, model: P200, catalog number: F123601 )
  7. PIPETMAN Classic pipet (Gilson, model: P1000, catalog number: F123602 )
  8. Homogenizer (MP Biomedicals, model: FastPrep-24TM 5G )
  9. Homogenizer adaptor (MP Biomedicals, model: CoolPrepTM 24x2mL )
  10. Vacuum manifold (QIAGEN, model: QIAvac 24 Plus )
  11. Heat block (VWR, catalog number: 75838-286 )
  12. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  13. Bioanalyzer (Agilent Technologies, model: 2100 Bioanalyzer )

Procedure

  1. Cell collection
    This protocol is designed for use on ~2.5 x 108 cells or less (e.g., 50 ml of culture at 5 x 106 cells/ml density) of liquid cultures of C. zofingiensis. See Figure 1 for example.


    Figure 1. Liquid cultures of C. zofingiensis. An example of liquid cultures of C. zofingiensis (SAG 211-14) suitable as input for this protocol. Here, cells were grown in Proteose medium (UTEX Culture Collection of Algae) supplemented with Chu’s medium (2 ml/L, UTEX Culture Collection of Algae) as described previously (Roth et al., 2017). The cultures were grown at 25 °C with shaking at 100-150 rpm in diurnal (16 h light, 8 h dark) conditions with a light intensity of 90-100 μmol photons m-2 sec-1.

    1. Transfer cell culture to a 50 ml Falcon tube.
    2. Spin for 5 min at 3,200 x g (RCF) at 4 °C. Immediately after the centrifuge comes to a stop, quickly remove the supernatant by decanting into a waste receptacle.
    3. Flash freeze tubes containing cell pellet in liquid N2.
    Optional: Store samples at -80 °C before further processing.

  2. Preparation
    1. Prepare glassware and metalware including spatula by washing with detergent and baking at 180 °C for 8 h.
    2. Clean bench and pipettes with RNaseZap or equivalent.
    3. Prepare fresh lysis buffer (see Recipes) just prior to starting according to the recipe.
    4. Pre-cool microfuge, centrifuge and buckets to 4 °C.
    5. Prepare buckets with crushed wet ice.
    6. Prepare buckets with crushed dry ice.
    7. Chill 100% EtOH on dry ice.
    8. Label Lysing Matrix D tubes and place on dry ice. 

  3. Cell lysis and RNA purification
    1. Transfer frozen sample tubes to the dry ice bucket.
    2. Take a clean, RNase-free spatula and thrust it up and down into the tube to break up the cell pellet. The tube should remain on dry ice during the process so that the cells remain frozen and prevent RNA degradation.
    3. Pipette 1 ml of cold 100% EtOH into the tube.
    4. Transfer the cell mass with spatula and EtOH with a pipette into a chilled Lysing Matrix D 2 ml tube.
    5. Spin for 3 min at 2,700 x g (RCF) at 4 °C. Decant supernatant.
    6. Place crushed dry ice in the homogenizer tray, transfer tubes to the homogenizer, and run for 60 sec at 6.0 m/sec. Repeat for an additional 60 sec.
    7. Add 1 ml of freshly prepared lysis buffer to the cell mass.
    8. Vortex for ~30 sec.
    9. Incubate for 3 min at RT.
    10. Spin for 3 min at 20,800 x g (RCF) at RT.
    11. Spin MaXtract tubes for 2 min at 1,500 x g (RCF) at RT.
    12. Add 10 ml of TRIzol to the MaXtract tube.
    13. Transfer 1 ml of supernatant to the MaXtract tube with TRIzol.
    14. Incubate for 3 min at RT.
    15. Add 2 ml of chloroform:isoamyl alcohol, and shake vigorously for 30 sec.
    16. Incubate for 5 min at RT.
    17. Centrifuge for 5 min at 1,500 x g (RCF) at RT.
    18. Add 10 ml of cold 100% EtOH to a labeled 50 ml conical tube for each sample.
    19. Decant the aqueous phase of the MaXtract tube (~6-7 ml) into the 50 ml conical tube with EtOH.
    20. Invert the tube several times to mix, and then place on wet ice.
    21. Working with one sample at a time, while the others remain on ice:
      1. Turn on the vacuum. Place a QIAGEN miRNeasy mini-column on the vacuum manifold with a fresh VacConnector.
      2. Using a 10 ml serological pipette, load the lysate/EtOH mixture onto the miRNeasy mini column until all liquid has passed through the membrane.
      3. Close vacuum and place column back in its 2 ml collection tube and put on ice. 
    22. Put column back on vacuum manifold and add 350 µl QIAGEN buffer RWT. Open vacuum until all buffer has passed through the column.
    23. Return the column to the collection tube.
    24. Just prior to using, prepare fresh DNase solution from the QIAGEN RNase-free DNase Set. Combine sufficient DNase (10 µl per column) and QIAGEN buffer RDD (70 µl per column) for all samples. Mix gently by inverting the tube 3-4 times.
      Note: Do NOT vortex the DNase!
    25. Add 80 µl DNase/RDD mixture from the previous step to each column.
    26. Incubate tubes for 15 min in a 30 °C heat block.
    27. Place columns back on the vacuum manifold.
    28. Add 350 µl QIAGEN buffer RWT to each column.
    29. Open vacuum until all RWT buffer has passed through the columns.
    30. Wash columns with QIAGEN buffer RPE buffer twice as follows:
      1. Add 500 µl of RPE to each column.
      2. Close the lid and rotate the column gently to rinse the walls of the column.
      3. Place columns back on the vacuum manifold.
      4. Open vacuum until all RPE buffer has passed through the columns.
      5. Repeat.
    31. Carefully remove the column from the collection tube to avoid carryover of ethanol and place it in a new 2 ml collection tube.
    32. Spin for 1 min at maximum speed (≥ 10,000 x g (RCF)) at RT.
    33. Place the column in a new, labeled 1.5 ml collection tube.
    34. Add 50 µl RNase-free dH2O directly to the spin column membrane.
    35. Spin for 1 min at maximum speed (≥ 10,000 x g (RCF)) at RT to elute the RNA.
    36. Repeat with an additional 50 µl RNase-free dH2O (~90 µl eluate in total).
    37. Add 270 µl RNase-free dH2O.
    38. Add 40 µl 3 M NaOAc at pH 8. Recap and invert the tube to mix.
    39. Add 1,000 µl ice-cold 100% EtOH. Recap and invert the tube to mix.
    40. Incubate for 30 min at -20 °C.
    41. Spin for 30 min at 4 °C at maximum speed (≥ 10,000 x g (RCF)).
    42. Without disturbing the pellet, gently decant supernatant.
    43. Wash with 500 µl RT 70% EtOH.
    44. Without disturbing the pellet, gently decant supernatant. Remove remaining liquid with a Pipetman.
    45. Air dry the pellet for 5-10 min at RT.
    46. Resuspend the pellet in 60 µl RNase-free dH2O.

  4. Quality control
    1. Determine the optical absorbance by NanoDrop spectrophotometer. Use 1 µl of dH2O to blank the NanoDrop.
    2. Determine the optical absorbance at 260, 230, and 280 nm for 1 µl of each sample. Assay the RNA quality by microfluidic electrophoresis on a Bioanalyzer.
      1. Dilute 1 µl of each sample with 9 µl of RNase-free water.
      2. Following the manufacturer’s protocol, load 1 µl of the 10x diluted RNA into a well of an RNA 6000 Nano chip and assay with the Eukaryote total RNA program.
        Note: Alternatively, an Agilent Tape Station may be used.

Data analysis

  1. Review the NanoDrop spectrophotometer data
    1. The A260/A280 ratio should be approximately 2.1. Discard sample if ratio is < 2.0 or > 2.2.
    2. The A260/A230 ratio should be approximately 2.1-2.4. Discard sample if ratio is < 2.0.
  2. Review the Bioanalyzer report
    1. There should be prominent peaks at ~1,800 nt and ~3,200 nt for the 18S and 28S, respectively (Figure 2).
    2. The RIN value calculated by the Agilent software should be > 8. It is recommended that samples with RIN < 7 should be discarded.
      Note: RIN values calculated by the software may be lower than similar quality RNA from other species due to abundant plastid rRNA in C. zofingiensis preparations. 


      Figure 2. Electropherogram of C. zofingiensis RNA. Total RNA from C. zofingiensis was prepared by the accompanying protocol and ran on an Agilent 2100 BioAnalyzer with a Eukaryote Total RNA Pico chip.

Notes

RNA is extremely sensitive to damage by RNase. Careful laboratory hygiene is crucial for minimizing this risk. Additionally, cells must be kept frozen until lysis buffer is added to prevent RNA degradation. When preparing many RNA samples, some may not satisfy the quality control criteria specified in the Data analysis section. It may be prudent to prepare more replicate samples than your experiment requires so that some may be discarded at the end of this protocol without compromising the overall study.

Recipes

  1. Lysis buffer

    Volumes given are for 10 ml. Adjust up or down as needed
    Add the SDS and Proteinase K in just before use

Acknowledgments

This protocol has been adapted from a previously published paper (Roth et al., 2017). This work was supported by the Agriculture and Food Research Initiative Competitive Grant No. 2013-67012-21272 from the USDA National Institute of Food and Agriculture (to M.S.R), and by a cooperative agreement with the US Department of Energy Office of Science, Office of Biological and Environmental Research program under Award DE-FC02-02ER63421. The authors declare no conflicts of interest or competing interests.

References

  1. Breuer, G., Lamers, P. P., Martens, D. E., Draaisma, R. B. and Wijffels, R. H. (2012). The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour Technol 124: 217-226.
  2. Dönz, O.C. (1934). Chlorella zofingiensis, eine neue Bodenalge. Ber Schweiz Bot Ges 43: 127-131.
  3. Fučíková, K. and Lewis, L. A. (2012). Intersection of Chlorella, Muriella and Bracteacoccus: Resurrecting the genus Chromochloris Kol et Chodat (Chlorophyceae, Chlorophyta). Fottea 12: 83-93.
  4. Liu, J., Mao, X., Zhou, W. and Guarnieri, M. T. (2016). Simultaneous production of triacylglycerol and high-value carotenoids by the astaxanthin-producing oleaginous green microalga Chlorella zofingiensis. Bioresour Technol 214: 319-327.
  5. Mulders, K. J. M., Janssen, J. H., Martens, D. E., Wijffels, R. H. and Lamers, P. P. (2014). Effect of biomass concentration on secondary carotenoids and triacylglycerol (TAG) accumulation in nitrogen-depleted Chlorella zofingiensis. Algal Res 6: 8-16.
  6. Roth, M. S., Cokus, S. J., Gallaher, S. D., Walter, A., Lopez, D., Erickson, E., Endelman, B., Westcott, D., Larabell, C. A., Merchant, S. S., Pellegrini, M. and Niyogi, K. K. (2017). Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production. Proc Natl Acad Sci U S A 114(21): E4296-E4305.

简介

Chromochloris zofingiensis 是一种单细胞绿藻,是生物燃料生产,酮类胡萝卜素生物合成和代谢等领域研究的新兴模式物种。 最近获得的高质量基因组组装便于系统级分析,如RNA-Seq。 然而,这种藻类的细胞具有坚韧的细胞壁,这对于RNA纯化来说是一个挑战。 该方案专门设计用于破坏细胞壁并分离适合RNA-Seq研究的高质量RNA。

【背景】Chromochloris zofingiensis 是一种来自叶绿体谱系的小型单细胞绿藻(Dönz,1934)。此前,该物种在文献中已被描述为属于Muriella ,小球藻和 Mychonastes (Fučíková和Lewis,2012)。 C中有强烈的经济利益。因为它能够生产大量用于生物燃料的脂质,并且显示有望成为具有商业价值的营养制品虾青素的来源(Breuer等人,2012; Mulders等人。,2014; Liu et。,2016)。最近,发布了高质量的染色体水平的基因组组装和附带的注释,这有助于系统水平分析,如RNA-Seq(Roth等人,2017)。以下方案被设计为从液体培养物中产生高度纯化的总RNA,包括miRNA。 zofingiensis 适合RNA-Seq。 ℃。 zofingiensis 细胞受坚固细胞壁的保护,该细胞壁被设计为穿透。该方案的起始材料可以高达2.5×10 8个总细胞。该方案应通过标准试剂盒(如Illumina TruSeq Stranded Total RNA试剂盒)产生至少20μg适合制备RNA-Seq文库的高度纯化的RNA。

关键字:佐夫色绿藻, 藻类, RNA纯化, RNA-Seq, 细胞壁

材料和试剂

  1. 50ml Falcon管(Corning,Falcon ,目录号:352070)
  2. 血清移液管,10毫升(Fisher Scientific,目录号:13-676-10J)
  3. Lysing Matrix D管(2 ml)(MP Biomedicals,产品目录号:116913050)
  4. 锥形末端,金属刮刀(Fisher Scientific,目录号:14-374)
  5. VacConnectors(QIAGEN,产品目录号:19407)
  6. ℃。 zofingiensis (SAG,目录号:211-14)
  7. 蛋白培养基(UTEX培养藻类,蛋白培养基)
  8. 楚氏培养基(UTEX藻类培养物,楚氏培养基)
  9. 液氮(LN 2)(各种)
  10. 湿冰(各种)
  11. 干冰(各种)
  12. RNaseZap(250ml喷雾瓶)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9780)。
  13. 无RNase乙醇(乙醇)(100%和70%)(各种)
  14. TRIzol(100ml)(Thermo Fisher Scientific,Invitrogen TM,目录号:15596026)
  15. 氯仿:异戊醇(24:1)(Sigma-Aldrich,目录号:C0549-1PT)
  16. MaXtract高密度(100 x 15毫升)(QIAGEN,目录号:129065)
  17. miRNeasy Mini Kit(50)(QIAGEN,目录号:217004),其中包括:
    1. QIAGEN miRNeasy迷你色谱柱QIAGEN miRNeasy迷你色谱柱
    2. 1.5和2毫升收集管
    3. QIAGEN缓冲区RWT颜色:#333333; font-family:&amp; font-size:14px; font-style:normal; font-variant:normal ;字型重量:400;字母间距:正常;孤儿:2;文本对齐:左;文字修饰:无;文本缩进:0像素;文字变换:无; -webkit文本行程宽度: 0像素;空白:正常;字间距:0像素;” />
    4. QIAGEN缓冲区RPE
    5. 无RNase水
  18. RNase-Free DNase Set(50个样品)(QIAGEN,目录号:79254),其中包括:
    1. 无RNA酶的DNA酶I
    2. QIAGEN缓冲区RDD
  19. 3M醋酸钠(NaOAc),pH = 8,无RNase(各种)
  20. 1M Tris盐酸(Tris-HCl),pH = 8,无RNase(各种)
  21. 5 M氯化钠(NaCl),无RNase(各种)
  22. 0.5M乙二胺四乙酸二钠盐二水合物(EDTA),pH = 8,无RNase(各种)
  23. 10%十二烷基硫酸钠(SDS)(各种)
  24. 20mg / ml蛋白酶K(Fisher Scientific,目录号:BP1700-100)
  25. 安捷伦RNA 6000纳米试剂盒(Agilent Technologies,目录号:5067-1511)
  26. 裂解缓冲液(见食谱)

设备

  1. 冷冻离心机(Eppendorf,型号:5810 R)
  2. 冷冻微型离心机(Eppendorf,型号:5424 R)
  3. 洁净工作台
  4. 冰桶(康宁,目录编号:432122)
  5. PIPETMAN Classic吸管(Gilson,型号:P20,目录号:F123600)
  6. PIPETMAN Classic吸管(Gilson,型号:P200,目录号:F123601)
  7. PIPETMAN经典吸管(Gilson,型号:P1000,目录号:F123602)
  8. 匀浆器(MP Biomedicals,型号:FastPrep-24 TM 5G)
  9. 匀浆器适配器(MP Biomedicals,型号:CoolPrep TM 24x2mL)
  10. 真空歧管(QIAGEN,型号:QIAvac 24 Plus)
  11. 加热块(VWR,目录号:75838-286)
  12. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000)
  13. 生物分析仪(安捷伦科技,型号:2100生物分析仪)

程序

  1. 细胞收集
    该协议设计用于约2.5×10 8个细胞或更少(例如50毫升5×10 6细胞/孔的培养物) ml密度)的液体培养物C.zofingiensis 。例如见图1。


    图1. C的液体培养物。 zofingiensis 。 C的液体培养物的例子。 zofingiensis (SAG 211-14)适合作为本协议的输入。在此,如前所述(Roth等人,2017),使细胞在补充有Chu's培养基(2ml / L,UTEX Culture Collection of Algae)的Proteose培养基(UTEX Culture Collection of Algae)中生长。培养物在25°C下在昼夜(16小时光照,8小时黑暗)条件下以100-150rpm振荡生长,光照强度为90-100μmol光子m 2 -2秒 -1 。

    1. 将细胞培养物转移到50ml Falcon管中。
    2. 在4℃下以3,200×g(RCF)旋转5分钟。立即在离心机停止后,通过倾倒入废液容器快速移除上清液。
    3. 闪烁冷冻含有液体N 2下的细胞团的管。
    可选:在进一步处理之前将样品存储在-80°C。

  2. 制备

    1. 使用洗涤剂清洗并在180°C下烘烤8小时,准备玻璃器皿和金属器具(包括刮铲)。
    2. 使用RNaseZap或同等产品清洁工作台和移液器。

    3. 根据配方准备新鲜的裂解缓冲液(参见配方)。
    4. 预冷微量离心机,离心机和水桶至4°C。

    5. 用碎冰块准备桶。

    6. 用碎冰块准备桶。

    7. 在干冰上冷藏100%乙醇。

    8. 标签Lysing Matrix D管并放置在干冰上。&nbsp;

  3. 细胞裂解和RNA纯化
    1. 将冷冻样品管转移到干冰桶中。
    2. 取一个干净,不含RNase的刮刀,上下推入管内,打碎细胞团。在这个过程中,管子应该保持在干冰上,以便细胞保持冷冻并防止RNA降解。
    3. 吸取1毫升冷的100%乙醇到管中。
    4. 用移液管将刮刀和EtOH转移到冷冻的Lysing Matrix D 2 ml试管中。
    5. 在4℃下以2,700×g(RCF)旋转3分钟。澄清上清液。
    6. 将破碎的干冰置于均质器托盘中,将管子输送至均化器,并以6.0米/秒运行60秒。重复60秒。

    7. 添加1毫升新鲜制备的裂解缓冲液
    8. 涡旋约30秒。
    9. 在室温孵育3分钟。

    10. 在20,800xg(RCF)旋转3分钟
    11. 在室温下1,500 x g(RCF)旋转MaXtract管2分钟。

    12. 添加10毫升TRIzol到MaXtract管
    13. 用TRIzol将1毫升上清液转移到MaXtract管中。
    14. 在室温孵育3分钟。
    15. 加入2毫升氯仿:异戊醇,剧烈振荡30秒。
    16. 在RT孵育5分钟。
    17. 在室温下以1,500×g(RCF)离心5分钟。

    18. 每个样品加入10毫升冷的100%乙醇到标记的50毫升锥形管中。
    19. 使用EtOH将MaXtract管的水相(约6-7ml)倒入50ml锥形管中。
    20. 将管子翻转数次混合,然后放在湿冰上。
    21. 一次处理一个样本,而其他样本一直处于冻结状态:
      1. 打开真空。用新鲜的VacConnector将QIAGEN miRNeasy mini-column放置在真空腔体上。
      2. 使用10 ml血清移液管,将裂解液/ EtOH混合物加载到miRNeasy mini柱上,直到所有液体都通过膜。
      3. 关闭真空并将色谱柱放回其2毫升收集管中并置于冰上。&nbsp;
    22. 将色谱柱放回真空管,加入350μlQIAGEN缓冲液RWT。打开真空,直到所有缓冲液都通过色谱柱。
    23. 将色谱柱返回收集管。
    24. 在使用前,请准备QIAGEN RNase-free DNase Set中的新鲜DNase溶液。将足够的DNA酶(每个柱10μl)和QIAGEN缓冲液RDD(每个柱70μl)合并为所有样品。通过颠倒管子3-4次轻轻混合。
      注意:不要旋转DNase!

    25. 在上一步中添加80μlDNase / RDD混合物到每个柱子。

    26. 在30°C的加热块中孵育管15分钟
    27. 将色谱柱放回真空歧管。

    28. 每个色谱柱加入350μlQIAGEN缓冲液RWT
    29. 打开真空,直到所有RWT缓冲液通过色谱柱。
    30. 用QIAGEN缓冲液RPE缓冲液清洗两次柱子如下:

      1. 每个色谱柱添加500μlRPE
      2. 关闭盖子并轻轻旋转柱子以冲洗柱子的壁。
      3. 将色谱柱放回真空歧管。
      4. 打开真空,直到所有RPE缓冲液通过色谱柱。
      5. 重复。
    31. 小心地从收集管中取出柱子以避免乙醇遗留,并将其放入新的2ml收集管中。
    32. 在室温下以最大速度旋转1分钟(≥10,000 em x g(RCF))。
    33. 将色谱柱放入一个新的1.5 ml收集管中。

    34. 。将50μl不含RNase的dH 2 O直接加入离心柱膜。
    35. 在室温下以最大速度旋转1分钟(≥1,000em x g(RCF))以洗脱RNA。
    36. 用另外的50μl无RNA酶的dH 2 O(约90μl洗脱液)重复。
    37. 加入270μlRNase-free dH sub 2 O。

    38. 加入40μl3 M NaOAc(pH 8)。重新收集并倒置管混合。
    39. 加入1,000μl冰冷的100%EtOH。回顾并翻转管混合。

    40. 在-20°C孵育30分钟

    41. 在4°C以最大速度(≥10,000 em x g (RCF))旋转30分钟。

    42. 在不干扰颗粒的情况下,轻轻滗析上清液。
    43. 用500μlRT 70%乙醇洗涤。
    44. 不干扰沉淀,轻轻倒出上清液。用移液器清除残留的液体。

    45. 在室温下空气干燥颗粒5-10分钟

    46. 用60μlRNase-free dH 2 O重悬沉淀
  4. 质量控制
    1. 用NanoDrop分光光度计测定吸光度。使用1μl的dH sub 2 O去空白NanoDrop。
    2. 确定每个样品1μl的260,230和280nm的吸光度。通过生物分析仪上的微流体电泳测定RNA质量。
      1. 用9μl无RNase的水稀释每个样品1μl。
      2. 遵循制造商的协议,将1μl10倍稀释的RNA加载到RNA 6000纳米芯片的孔中,并使用真核生物总RNA程序进行测定。
        注意:或者,也可以使用Agilent Tape Station。

数据分析

  1. 查看NanoDrop分光光度计数据
    1. A 260 / A 280比率应当约为2.1。如果比例为&lt; 2.0或&gt; 2.2。
    2. A 260 / A 230比率应当约为2.1-2.4。如果比例为&lt; 2.0。
  2. 查看生物分析仪报告
    1. 18S和28S应该在〜1800 nt和〜3200 nt处有明显的峰值(图2)。
    2. 由安捷伦软件计算的RIN值应该> 8.建议使用RIN <应该丢弃7个。
      注:由软件计算的RIN值可能低于来自其他物种的类似质量的RNA,这是由于在C.zofingiensis制剂中存在丰富的质体rRNA。&nbsp;


      图2. C. zofingiensis RNA的电泳图。 C 的总RNA。 zofingiensis 由相应的方案制备,并使用Agilent 2100 BioAnalyzer与真核生物总RNA Pico芯片一起运行。

笔记

RNA对RNA酶造成的损伤非常敏感。仔细的实验室卫生对于降低这种风险至关重要。另外,细胞必须保持冷冻直至加入裂解缓冲液以防止RNA降解。准备许多RNA样品时,有些可能不符合数据分析部分中指定的质量控制标准。准备比实验要求更多的重复样本可能是明智的,以便在本协议结束时可以丢弃一些样本而不影响整体研究。

食谱

  1. 裂解缓冲液

    给定的体积为10毫升。根据需要调整或调低
    在使用前添加SDS和蛋白酶K

致谢

该协议已被改编自以前发表的论文(Roth等人,2017年)。这项工作得到了美国农业部国家食品与农业研究所(MSR)的农业与食品研究计划竞争性拨款号2013-67012-21272以及与美国能源部科学办公室的合作协议的支持生物和环境研究项目获奖DE-FC02-02ER63421。作者声明不存在利益冲突或利益冲突。

参考

  1. Breuer,G.,Lamers,P. P.,Martens,D. E.,Draaisma,R.B。和Wijffels,R.H。(2012)。 氮饥饿对9种微藻菌株中三酰甘油积累动态的影响。
  2. Dönz,O.C. (1934年)。 Chlorella zofingiensis,eine neue Bodenalge。 Ber Schweiz Bot Ges 43:127-131。
  3. Fučíková,K.和Lewis,L. A.(2012)。 小球藻交集, Muriella 和 Bracteacoccus :复活染色体属Chromochloris Kol et Chodat(Chlorophyceae,Chlorophyta)。 Fottea 12:83-93。
  4. Liu,J.,Mao,X.,Zhou,W.和Guarnieri,M. T.(2016)。 虾青素产油绿藻微藻同时生产三酰甘油和高价值类胡萝卜素小球藻zofingiensis 。 Bioresour Technol 214:319-327。
  5. Mulders,K.J.M.,Janssen,J.H.,Martens,D.E.,Wijffels,R.H.和Lamers,P.P。(2014)。 生物量浓度对缺氮小球藻中次生类胡萝卜素和三酰甘油(TAG)积累的影响zofingiensis。 Algal Res 6:8-16。
  6. Roth,MS,Cokus,SJ,Gallaher,SD,Walter,A.,Lopez,D.,Erickson,E.,Endelman,B.,Westcott,D.,Larabell,CA,Merchant,SS,Pellegrini, Niyogi,KK(2017)。 染色体水平的基因组组装和绿藻Chromochloris zofingiensis 的转录组照亮 虾青素生产。 美国国家科学院院刊114(21):E4296-E4305。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gallaher, S. D. and Roth, M. S. (2018). RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis. Bio-protocol 8(7): e2793. DOI: 10.21769/BioProtoc.2793.
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