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Jan 2020

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Candida albicans Culture, Cell Harvesting, and Total RNA Extraction
白念珠菌培养、细胞收获和总RNA提取   

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Abstract

Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the form of the tough cell wall made up of many different components such as chitin that are resistant to many common mammalian or bacterial cell lysis methods. Typical in vitro C. albicans cell harvesting methods can be time consuming and expensive if many samples are being processed with multiple opportunities for product loss or sample variation. Harvesting cells via vacuum filtration rather than centrifugation cuts down on time before the cells are frozen and therefore the available time for the RNA expression profile to change. Vacuum filtration is preferred for C. albicans for two main reasons: cell lysis is faster on non-pelleted cells due to increased exposed surface area, and filamentous cells are difficult to pellet in the first place unlike yeast or bacterial cells. Using mechanical cell lysis, by way of zirconia/silica beads, cuts down on time for processing as well as overall cost compared to enzymatic treatments. Overall, this method is a fast, efficient, and high-yield way to extract total RNA from in vitro cultures of C. albicans.

Keywords: Candida albicans (白念珠菌), RNA extraction (RNA提取), Cell harvest (细胞收获), Fungal transcription (真菌转录), RNA isolation (RNA分离)

Background

The need for fast, reproducible, and efficient RNA extraction techniques has grown significantly over the last several years due to the steady increase in use of RNA sequencing and other expression analysis techniques that have become more affordable and faster with improvements in sequencing technology. There are many different kits and protocols out there from various companies and labs that attempt to meet this need. However, methods that are built specifically for one type of fungi may not be usable for another, and kit platforms can often fall short by way of being too broad in their application. Here we describe a cell culture, harvest, and RNA extraction method for the pathogenic fungus, Candida albicans, that utilizes a combination of techniques to give both high yield and high-quality RNA in a consistent and efficient manner.


One of the main attributes unique to this approach is the harvest of cells via vacuum filtration rather than by centrifugation. Centrifugation of a 25 ml culture of filamentous cells, as is used in this protocol, must be done over a period of 5 min in order to pellet the cells enough to aspirate the growth media. This additional time before freezing the cells and halting cellular processes opens the door for unwanted transcriptional changes. Previous studies have shown that yeast cells can alter their expression profiles well within the 5 min that is needed to spin cells down (Dikicioglu et al., 2011). It is then of critical importance to shorten the time between the incubation/growth period and when the cells are frozen, and vacuum filtration serves this purpose well. It can take anywhere from 5-10 min between incubation and freezing when using centrifugation, but that time decreases to 1-2 min using vacuum filtration with the most time-consuming step being transporting the samples from the bench to the freezer. Not only does this saved time cut down on transcriptional variation, but it also allows for more samples to be processed in the same amount of time increasing overall throughput for this method.


The second variation in this this approach that differs from other methods is the use of zirconia/silica beads in combination with a lysis buffer for cell disruption as opposed to enzymatic or lysis buffer only methods. Mechanical cell disruption via bead beating has been shown to significantly increase RNA yields in C. albicans, compared to vertical vortexing without beads in lysis buffer alone (Rodríguez and Vaneechoutte, 2019). Zirconia/silica beads have a higher density than the typical glass beads used in bacterial (3.7 g/cm3 and 2.5 g/cm3 respectively) which increases their efficiency at rupturing the tough fungal cell walls. An additional benefit of mechanical cell disruption is that enzymatic methods such as zymolyase digestion have been shown previously to alter RNA expression profiles of the sample being assayed by activating stress response pathways thereby confounding the downstream analysis (Suzuki and Iwahashi, 2013).


Overall, by utilizing mechanical cell disruption and vacuum filtration combined with the commercially available Qiagen RNEasy MiniKit, this technique represents a fast, and efficient method for cell harvesting and RNA extraction saving time and giving more consistent and reliable transcriptional data.


Materials and Reagents

  1. 15 ml polypropylene culture tubes (VWR, catalog number: 82050-274 , item #187262)

    Note: Can be from any source.

  2. 25 ml serological pipettes (VWR, catalog number: 89130-912 )

    Note: Can be from any source.

  3. 50 ml conical screw cap tube (Fisher Scientific, catalog number: 0553913)

    Note: Can be from any source.

  4. 1.5 ml screw cap tubes (Fisher Scientific, catalog number: 1415-8700 )

    Note: Can be from any source.

  5. RNase-free 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 14-666-319 )

    Note: Can be from any source.

  6. 250 ml 0.1 µm PES membrane vacuum filtration unit (Fisher Scientific, catalog number: 09741201)

    Note: Can be from any source.

  7. Cuvettes PS Semi-micro (VWR, catalog number 9700-586 )

    Note: This is dependent upon the method of measuring the OD600 of cell cultures to be used.

  8. Uvette 220-1600 nm (Eppendorf, catalog number: 952010051 )

    Note: This is dependent upon the method of quantifying purified RNA.

  9. 0.5 mm zirconia/silica disruption beads (Research Products International, catalog number: 9834 )

    Follow manufacturer instructions to sterilize and eliminate nucleic acid contamination and store at -20 °C

  10. MF-MilliporeTM 0.45 µm, 47 mm diameter gridded filter membrane (Merk Millipore Ltd., MF-MilliporeTM, catalog number: HAWG04700 )

  11. Relevant C. albicans strains (SC5314 and an SC5314 derived efg1Δ::HIS1 mutant strain constructed by the authors were used in this example)

  12. Yeast extract (BD, BactoTM, catalog number: 212750 )

  13. Peptone (BD, BactoTM, catalog number: 211677 )

  14. Dextrose (Sigma Life Science, catalog number: D9434-1KG )

  15. RPMI 1640 (Sigma-Aldrich, catalog number: R4130-10L ), store at 4 °C

  16. Fetal Bovine Serum Premium (Atlanta Biologicals, R&D Systems, catalog number: S11150H ), store at -20 °C

  17. Phenol:chloroform:isoamyl alcohol (Sigma Life Science, catalog number: 77617-100ml ), store at 4 °C

  18. Qiagen RNEasy Mini Kit (Qiagen, catalog number: 74104 )

  19. NaOH salt (Sigma-Aldrich, catalog number: 221465-500G )

    Note: Can be from any source.

  20. 100% pure Ethanol (Sigma-Aldrich, catalog number: E7023-1L )

    Note: Can be from any source.

  21. Deionized H2O (any source)

  22. β-mercaptoethanol (Sigma-Aldrich, catalog number: 444203-250mL )

    Note: Can be from any source.

  23. YPD liquid growth media (see Recipes)

  24. 10 N NaOH (see Recipes)

  25. RPMI + 10% FBS (see Recipes)

Equipment

  1. Glass 125 ml Erlenmeyer flasks (Fisher Scientific, catalog number: FB501125 )

    Note: Can be from any source.

  2. 30 °C incubator (any source)

  3. Rotator drum for overnight cultures (New Brunswick Scientific, model: TC-7 , catalog number: M1053-4004)

    Note: This is out of production but can be substituted for a similar rotator that can reach 60-70 rpm.

  4. Eppendorf BioPhotometer® D30 (Eppendorf, catalog number: 6133000010 )

    Note: This can be substituted for another spectrophotometer with similar capabilities.

  5. Shaking incubator 37 °C (New Brunswick Scientific, Eppendorf, model: I-26, catalog number: M1324-0000)

    Note: This can be substituted for another shaking incubator with similar capabilities.

  6. Microcentrifuge (Thermo Scientific, model: Sorvall Legend Micro 21, catalog number: 75002436)

    Note: This can be substituted for another microcentrifuge that can spin at ≥ 17,000 x g.

  7. 1-2 L sidearm Erlenmeyer flask (any source)

  8. MiniBeadBeater-16 (BioSpec Products, model: 607 )

    Note: The authors have not attempted this protocol with another type of bead beater, however a similar horizontal bead beater with similar specifications would most likely yield similar results.

  9. Millipore 47 mm glass base and stopper (Millipore, catalog number: XX1014702 )

  10. 50 ml conical tube rack (any source)

  11. -80 °C and -20 °C freezer (any source)

  12. 4 °C refrigerator or cold room (any source)

  13. Standard benchtop vortexer (any source)

  14. Forceps

Procedure

Note: This procedure is written with volumes and quantities for two strains, mutant and Wild Type (WT), with three replicates each for a total of six independent samples.

  1. Cell culture and harvesting

    1. Inoculate WT and mutant strains in 5 ml liquid YPD and incubate overnight at 30 °C with 60 rpm rotation.

    2. Pre-warm 200 ml RPMI + 10% FBS and empty sterile 125 ml Erlenmeyer flasks overnight (O/N) 37 °C, or for a minimum of 2 h prior to Step A3 with more time for larger volumes.

    3. Aliquot 25 ml pre-warmed RPMI + 10% FBS into each of the 6 pre-warmed 125 ml flasks and return to 37 °C incubator to stabilize temperature at least 90 min before first inoculation.

    4. Vortex O/N WT culture thoroughly, measure OD600 using Eppendorf BioPhotometer and Cuvettes PS Semi-micro, and inoculate first pre-warmed flask of RPMI + 10% FBS to a final OD600 of 0.2 from the O/N WT culture.

    5. Immediately transfer to I-26 air shaker incubator for 4 h at 37 °C and 225 rpm rotation. Replace O/C in 30 °C incubation.

    6. Wait 8-10 min between inoculation of replicates.

      Note: This gap is to allow for adequate time for cell harvesting between replicates. Time can be shortened or lengthened as needed to allow for working speed of person performing protocol.

    7. Repeat Steps A4-A6 for WT replicates 2 and 3 as well as mutant replicates 1-3.

    8. This leaves roughly 3 h until the first culture is ready to harvest. Take this opportunity to set up cell harvesting equipment.

      1. Connect 1-2 L sidearm flask to a vacuum line and trap and mount the Millipore 47 mm glass base and stopper for vacuum filtration in the top of the flask.

      2. Turn on vacuum and wash filter base 2x with dH2O and 2x with 70% EtOH to eliminate debris and allow filter base to dry before applying filter membrane.

      3. Chill 50 ml conical tube rack at -80 °C.

      4. Pre-chill and label 6 50 ml conical screw-cap tubes in ice.

    9. Just before 4-hour timepoint apply MF-MilliporeTM 0.45 µm, 47 mm diameter gridded filter membrane to filter base grid side up using sterile flat-blade forceps and turn on vacuum (Figure 1).



      Figure 1. Cell harvest filtration setup. Millipore 47 mm glass base and stopper inserted into sidearm flask and connected to vacuum line. Wash glass base thoroughly with water and 70% ethanol with vacuum running and let dry. Place MF-MilliporeTM 0.45 µm, 47 mm diameter gridded filter membrane onto glass base. In-line trap and filter are suggested to avoid carryover of waste.


    10. At 4 h, remove the WT replicate 1 flask from the shaker and swirl gently to dislodge cells stuck to the sides of the flask.

    11. Quickly, use a 25 ml serological pipette to transfer entire culture to filter membrane as fast as the vacuum will allow without overflowing the filter membrane.

    12. As soon as the liquid has been pulled off of the cells, transfer filter membrane to bottom of a pre-chilled 50 ml conical tube using sterile flat-blade forceps, and place immediately in -80 °C freezer.

    13. Clean the filter base as before with dH2O and 70% EtOH.

    14. Repeat Steps A9-A13 for each of the remaining WT and mutant replicates.

      Note: These steps are time sensitive. Shortening the time between when the cells are incubating to when they are frozen at -80 °C is critical for accurate expression data.

    15. Incubate cells at -80 °C for at least 1 h to ensure they are frozen before proceeding to RNA extraction.

      Note: Cells are stable at this stage for several days to weeks, so if many samples are needed, cell culturing and harvesting can be split up over multiple days prior to RNA extraction.

  2. RNA extraction and quantification

    1. Aliquot 300 µl 0.5 mm zirconia/silica disruption beads and 600 µl 25:24:1 phenol:chloroform:isoamyl alcohol to 1.5ml screw cap tubes and chill at 4 °C for at least 1 h (1 tube per cell culture replicate). Prepare fresh 600 µl RLT (Qiagen RNeasy Mini Kit) + 1% β-mercaptoethanol (BME) per sample and pre-chill in ice. Pre-chill 2 ml sterile dH2O per sample on ice. Pre-chill 1.5 ml microcentrifuge tubes (1 per sample) on ice.

      Note: Steps B2-B10 must be done at 4 °C. Keep materials on ice or work in a cold room.

    2. Remove 50 ml conical tube containing filter membrane and cells from -80 °C freezer and thaw on ice for 10 min.

    3. Wash cells off of filter membrane with 900 µl of chilled sterile dH2O and vortex at top speed for 30 s. Transfer suspension to chilled 1.5 ml microcentrifuge tube and place on ice.

    4. Wash filter a second time with an additional 900 µl of chilled sterile dH2O and vortex at top speed for 30 s. Transfer suspension to the same chilled 1.5 ml microcentrifuge tube and place on ice.

    5. Repeat Steps B3 and B4 for all samples using a new tube for each sample.

    6. Centrifuge in standard desktop microcentrifuge at max speed (≥ 17,100 x g) for 30 s and discard supernatant.

    7. Add 600 µl prepared RLT + 1% BME solution and resuspend by vortexing at max speed.

    8. Transfer 600 µl of cell suspension to chilled screwcap tubes with zirconia beads and phenol:chloroform:isoamyl alcohol.

    9. Bead beat for 3 min at 4 °C using the BioSpec MiniBeadBeater-16 Model 607 .

    10. Centrifuge cells at max speed for 8 min at 4 °C in standard desktop microcentrifuge.

    11. Transfer 550 µl of the aqueous layer to new RNase-free 1.5 ml microcentrifuge tube and add 550 µl 70% EtOH and mix by inverting 6 times.

      Note: If there is not 550 µl of aqueous solution, measure and transfer to the new tube and add an equal volume of 70% EtOH.

    12. Transfer up to 700 µl of sample to RNeasy spin column and centrifuge for 15 s at ≥ 8,000 x g. Discard flow-through. Reload column with remaining sample and spin again discarding flow-through.

      Note: All supplies referred to in Steps B12-B17 are provided in the Qiagen RNeasy Mini Kit Protocol for the Purification of Total RNA from Yeast. Steps B12-B16 of this protocol are the same as steps 2-5 of the Qiagen RNeasy Mini Kit protocol for Purification of Total RNA from Yeast.

    13. Add 700 µl Buffer RW1 to column and spin for 15 s at ≥ 8,000 x g. Discard flow-through.

    14. Add 500 µl Buffer RPE (with Ethanol added) to column and spin for 15 s at ≥ 8,000 x g. Discard flow-through.

    15. Repeat Step B14.

    16. Transfer spin column to a new collection tube and spin for 1 min at ≥ 8,000 x g. Discard flow-through.

    17. Transfer spin column to RNase-free 1.5 ml microcentrifuge tube (from kit). Add 40 µl RNase-free water to the column membrane and centrifuge for 1 min at ≥ 8,000 x g.

    18. Take 40µl eluate from Step B17 and re-apply to the spin column membrane and spin again for 1 min at ≥ 8,000 x g.

      Note: This is not strictly necessary but will increase overall concentration.

    19. Freeze samples immediately at -80 °C for storage.

    20. To quantify RNA and assess purity, prepare 1:500 dilution of total RNA and measure OD260 using Eppendorf BioPhotometer and Uvette 220-1,600 nm cuvettes.

      Notes:

      1. Average yield is typically over 2 µg/µl up to around 6 µg/µl. 260/280 and 260/230 values typically range between 2.0-2.2 and 2.5-2.5 respectively.

      2. Dilution and use of Uvettes may not be necessary depending on equipment available.

Notes

  1. This protocol is written to accommodate 6 samples (2 strains with 3 replicates each) but is scalable given that it can be stretched over multiple days while the harvested cells are frozen. It can also be adapted for whatever media conditions outside of RPMI + 10% FBS. However, with the addition of more replicates and strains it is recommended that a single large batch of media is made, if possible, ahead of time to eliminate batch effects on the transcriptional profile of the strains. For example, here we call for 200 ml of RPMI + 10% FBS for 6 samples which allows for 50ml extra. If 60 samples were needed instead, 1.5 L of RPMI + 10% FBS should be made up in one large batch then aliquoted into smaller units for use over several days.

  2. This protocol does not include a DNase treatment. This means that the final product may have some DNA contamination. The Qiagen RNeasy Mini Kit spin columns prevent most DNA carry-over by design, but residual DNA may remain. If DNA contamination must be avoided for downstream applications, DNase treatment may be done on the spin column between Steps B12 and B13 or after final elution, and a protocol for this can be found in the Qiagen RNeasy Mini Kit protocol.

Recipes

  1. YPD liquid growth media

    1. Dissolve 20 g dextrose, 20 g peptone, and 10 g yeast extract with 500 ml dH2O

    2. Bring volume to 1 L with dH2O

    3. Autoclave to sterilize

  2. 10 N NaOH

    1. Dissolve 40 g NaOH in 50 ml dH2O

    2. Bring volume up to 100 ml with dH2O

  3. RPMI+10% FBS

    1. Dissolve 3.28 g RPMI1640 powder in 180 ml dH2O

    2. Adjust pH to 7.4 using 10 N NaOH (approximately 180 µl)

    3. Sterilize RPMI solution by vacuum filtration

    4. Thaw Fetal Bovine Serum Premium (FBS) in 37 °C water bath

      Note: The FBS manufacturer recommends avoiding repeated freeze thaw cycles, so plan accordingly.

    5. Shake gently to resuspend any solids

    6. Add 20 ml thawed FBS to 200 ml RPMI solution and mix with gentle shaking to avoid foaming

    7. Store at 4 °C for up to a month

Acknowledgments

Work on C. albicans RNA in this lab is supported by NIH grants 1R21AI144878-01 and 1R01AI146103 (PI: Mitchell). This protocol evolved from protocols in use in the Mitchell lab since 1988.

Competing interests

The author declare that no competing interests exist.

References

  1. Rodríguez, A. and Vaneechoutte, M. (2019). Comparison of the efficiency of different cell lysis methods and different commercial methods for RNA extraction from Candida albicans stored in RNAlater. BMC Microbiol 19(94).
  2. Suzuki, T. and Iwahashi, Y. (2013). RNA preparation of Saccharomyces cerevisiae using the digestion method may give misleading results. Appl Biochem Biotechnol 169(5): 1620-1632.
  3. Dikicioglu, D., Karabekmez, E., Rash, B., Pir, P., Kirdar, B. and Oliver, S. G. (2011). How yeast re-programmes its transcriptional profile in response to different nutrient impulses. BMC Syst Biol 5148.

简介

[摘要]转录分析已成为生物学研究的基石,并且在最近十年中,随着廉价,高效测序技术的出现,对高产量,高效率的RNA提取技术存在着需求。真菌如人病原体白色念珠菌呈现出独特的障碍RNA纯化在坚韧细胞壁的形式作出许多不同的部件,例如几丁质的最多的是对许多常见的哺乳动物细胞或细菌细胞裂解的方法具有抗性。典型的体外白色念珠菌 如果要处理的样品很多,并且产品损失或样品变化的机会很多,则细胞采集方法可能既耗时又昂贵。通过真空过滤而不是离心收集细胞可以减少冷冻前的时间,因此可以改变RNA表达谱的可用时间。对于白色念珠菌而言,真空过滤是优选的,这主要有两个原因:由于暴露的表面积增加,非丸状细胞的细胞裂解速度更快,并且与酵母或细菌细胞不同,丝状细胞首先难以沉淀。与酶法处理相比,使用机械细胞裂解法(通过氧化锆/二氧化硅珠子)可减少加工时间以及总成本。总的来说,该方法是从白念珠菌的体外培养物中提取总RNA的快速,高效且高产率的方法。

[背景]需要快速,可重复的,高效的RNA提取技术已经显著在过去几年里,由于使用RNA测序等表达分析技术的稳步增长成长是变得更加经济实惠,并与测序技术的改进速度更快。各种公司和实验室提供了许多不同的工具包和协议,试图满足这一需求。但是,专门为一种真菌建立的方法可能不适用于另一种真菌,而试剂盒平台由于其应用范围太广,常常会不足。在这里,我们描述了一种细胞培养物,收获,和RNA提取方法为致病性真菌,白色念珠菌,其利用的技术的组合,得到以一致和有效的方式既高产率和高质量的RNA。

该方法独有的主要属性之一是通过真空过滤而不是离心收集细胞。一个25的离心米升丝状细胞的培养,如在此协议中使用,必须在一段5分钟以沉淀足够吸出生长培养基的细胞来完成。冻结细胞和停止细胞过程之前的这段额外时间为不希望的转录变化打开了大门。先前的研究表明,酵母细胞可以在旋转细胞所需的5分钟内很好地改变其表达谱(Dikicioglu等,2011 )。因此,至关重要的是缩短孵育/生长期与细胞冷冻之间的时间,真空过滤很好地达到了这一目的。使用离心时,孵育和冷冻之间可能需要5-10分钟之间的任何时间,但是使用真空过滤的时间会减少到1-2分钟,其中最耗时的步骤是将样品从工作台运送到冰箱。这样不仅节省了时间,减少了转录变异,而且还允许在相同的时间内处理更多样品,从而提高了该方法的整体通量。

与其他方法不同的是,此方法的第二个变化是将氧化锆/硅胶珠与裂解缓冲液结合使用以破坏细胞,这与酶法或仅裂解缓冲液的方法不同。与单独在裂解缓冲液中不使用珠子的垂直涡旋相比,通过珠子敲打造成的机械细胞破坏已显示出可显着提高白色念珠菌的RNA产量(Rodríguez和Vaneechoutte ,2019 )。氧化锆/二氧化硅珠的密度高于细菌中使用的典型玻璃珠(分别为3.7 g / cm 3和2.5 g / cm 3 ),这增加了其破坏坚韧的真菌细胞壁的效率。机械破碎细胞的一个额外的好处是,酶的方法,如酵母裂解酶消化先前已显示与样品的ALTER RNA表达谱通过激活应激反应途径从而混杂下游分析(所测定铃木和岩桥,2013 )。

总体而言,通过将机械细胞破碎和真空过滤与可商购的Qiagen RNEasy MiniKit结合使用,该技术代表了一种快速有效的细胞收获和RNA提取方法,可节省时间并提供更一致和可靠的转录数据。

关键字:白念珠菌, RNA提取, 细胞收获, 真菌转录, RNA分离

材料和试剂

1 15 ml聚丙烯培养管(VWR,目录号:82050-274,项#187262)
注意:可以来自任何来源。


2 25 ml血清移液管(VWR,目录号:89130-912)
注意:可以来自任何来源。


3 50 ml锥形螺旋盖管(Fisher Scientific,目录号:0553913)
注意:可以来自任何来源。


4 1.5 ml螺帽管(Fisher Scientific,目录号1415-8700)
注意:可以来自任何来源。


5 无RNase的1.5 ml离心管(Fisher Scientific公司,Ç atalog号:14-666-319)
注意:可以来自任何来源。


6 250 ml 0.1 µm PES膜真空过滤装置(Fisher Scientific,目录号:09741201)
注意:可以来自任何来源。


7 Cuvettes PS Semi-micro(VWR,目录号9700-586)
注意:这取决于要使用的细胞培养物的OD 600的测量方法。


8 Uvette 220-1600 nm(Eppendorf,目录号:952010051)
注意:这取决于定量纯化RNA的方法。


9 0.5毫米氧化锆/二氧化硅破碎珠(国际研究产品,目录号:9834)
按照制造商的说明进行灭菌和消除核酸污染,并在-20°C下保存


10 MF- Millipore TM 0.45 µm,直径47 mm的网格滤膜(Merk Millipore Ltd.,MF- Millipore TM ,目录号:HAWG04700)
11 相关的白色念珠菌菌株(本例中使用作者构建的SC5314和SC5314衍生的efg1Δ :: HIS1突变株)
12 酵母提取物(BD,Bacto TM ,目录号:212750)
13 蛋白ept (BD,Bacto TM ,目录号:211677)
14 葡萄糖(Sigma Life Science,目录号:D9434-1KG)
15 RPMI 1640(Sigma-Aldrich公司,目录号:R4130-10L),小号撕4 ℃下
16 胎牛血清的高级(亚特兰大生物制品,R&d系统,目录号:S11150H),小号撕在-20℃下
17 苯酚:氯仿:异戊醇(Sigma公司生命科学,目录号:77617-100ml),小号撕在4℃下
18 Qiagen RNEasy迷你套件(Qiagen,目录号:74104)
19 NaOH盐(Sigma-Aldrich,目录号:221465-500G)
注意:可以来自任何来源。


20 100%纯乙醇(Sigma-Aldrich,目录号:E7023-1L )
注意:可以来自任何来源。


21 去离子H 2 O(任何来源)
22 β-巯基乙醇(Sigma-Aldrich,目录号:444203-250mL)
注意:可以来自任何来源。


23 YPD液体生长培养基(请参阅食谱)
24 10 N NaOH(请参阅食谱)
25 RPMI + 10%FBS(请参阅食谱)
 


设备


 1 125毫升玻璃烧瓶(Fisher Scientific,目录号:FB501125)
注意:可以来自任何来源。


2 30°C培养箱(任何来源)
3 旋转鼓过夜培养物(新不伦瑞克科学,米Odel等:TC-7,目录号:M1053-4004)
注意:此产品已停产,但可以替代可达到60-70 rpm的类似转子。


4 Eppendorf BioPhotometer生物分光光度计® D30仪(Eppendorf,目录号:6133000010)
注意:这可以替代另一台具有类似功能的分光光度计。


5 摇动培养箱37°C(New Brunswick Scientific,Eppendorf,型号:I-26,货号:M1324-0000)
注意:这可以替代另一台具有类似功能的振荡培养箱。


6 微量离心机(Thermo Scientific ,型号:Sorvall Legend Micro 21,目录号:75002436)
注意:这可以取代另一微量即能在≥旋转17000×g离心。


7 1-2升侧臂锥形烧瓶(任何来源)
8 MiniBeadBeater-16(BioSpec产品,型号:607)
注:牛逼,他的作者并没有试图将该协议与其他类型的玻珠打击的,但是具有类似规格类似的水平玻珠打击将最有可能产生类似的结果。


9 Millipore 47毫米玻璃底座和塞子(Millipore,目录号:XX1014702)
10 50 ml锥形管架(任何来源)
11 -80 °C和-20°C冰箱(任何来源)
12 4°C冰箱或冷藏室(任何来源)
13 标准台式涡旋仪(任何来源)
14 钳子
 


程序


 


注意:此程序以突变体和野生型(WT)两种菌株的体积和数量编写,每组三个重复,共六个独立样品。


 


A 细胞培养和收获
1.在5 ml液体YPD中接种WT和突变菌株,并在30°C旋转60 rpm孵育过夜。      


2.预热200 ml RPMI + 10%FBS并在125°C的无菌无菌锥形瓶中在37°C下过夜(O / N),或在步骤A3之前最少放置2 h,然后花更多的时间进行大体积处理。      


3.将25 ml预热的RPMI + 10%FBS分装到6个预热的125 ml烧瓶中的每一个中,并在首次接种之前至少回到90 °C的恒温箱,以稳定温度90分钟。      


4.彻底涡旋O / N WT培养,使用Eppendorf BioPhotometer和Cuvettes PS Semi-micro测量OD600 ,并从O / N WT培养物中接种第一个预保温的RPMI + 10%FBS烧瓶,使最终OD600为0.2。      


5.立即转移到I-26空气振荡器培养箱中,在37°C和225 rpm旋转下保持4 h。在30°C孵育中替换O / C。      


6.重复接种之间等待8-10分钟。      


注意:此间隙是为了留出足够的时间在两次重复之间进行细胞收获。可以根据需要缩短或延长时间,以允许执行协议的人员的工作速度。


7.对于WT重复2和3以及突变重复1-3,重复步骤A4-A6 。      


8.这大约需要3个小时,直到第一个培养物准备好收获。借此机会来建立细胞收集设备。      


a 将1-2 L侧臂烧瓶连接到真空管线,并收集并安装Millipore 47毫米玻璃底座和塞子,以便在烧瓶顶部进行真空过滤。
b 开启真空并用dH 2 O洗涤滤网2次,并用70%乙醇洗涤2次,以清除碎屑并让滤网干燥,然后再使用滤膜。
c 在-80°C下冷却50 ml锥形管架。
d 在冰中预冷并标记6个50 ml锥形螺口管。
9.在4小时的时间点之前,使用无菌平口钳将MF- Millipore TM 0.45 µm,直径47 mm的网格状滤膜朝上过滤,使基础网格面朝上,并打开真空(图1)。      


 






图1.细胞收集过滤设置。将Millipore 47毫米玻璃底座和塞子插入侧臂烧瓶中并连接到真空管线。在真空运行下,用水和70%乙醇彻底清洗玻璃基底,并使其干燥。将MF- Millipore TM 0.45 µm,直径47 mm的网格滤膜放在玻璃底座上。建议使用在线捕集器和过滤器,以免浪费。


 


10.在4 h时,从振荡器中取出WT复制1烧瓶,并轻轻旋转以移出粘在烧瓶侧面的细胞。   


11.迅速用25 ml血清移液管将整个培养物转移至滤膜,速度应尽可能快,而不会使真空膜溢出。   


12.一旦液体从细胞中移出,请使用无菌平刃镊子将滤膜转移至预冷的50 ml锥形管的底部,并立即置于-80°C的冰箱中。   


13.如前所述,用dH 2 O和70%的乙醇清洗滤清器底座。   


14.对于其余的WT和突变体重复序列,重复步骤A9-A13。   


注意:这些步骤是时间敏感的。对于精确的表达数据而言,缩短细胞孵育至-80°C冷冻之间的时间至关重要。


15.将细胞在-80°C下孵育至少1 h,以确保将其冷冻后再进行RNA提取。   


注意:细胞在此阶段可以稳定几天到几周,因此,如果需要大量样品,则可以在提取RNA之前的数天内将细胞培养和收获分离。


 


B RNA提取和定量
1.将30 0 µl 0.5 mm氧化锆/二氧化硅破裂珠和600 µl 25:24:1苯酚:氯仿:异戊醇等分装到1.5ml螺帽管中,并在4 °C冷却至少1 h(每个细胞培养1个管)复制)。每个样品准备新鲜的600 µl RLT(Qiagen RNeasy Mini试剂盒)+ 1%β-巯基乙醇(BME),并在冰中预冷。预先冷却2毫升无菌卫生署2 ö每在冰上的样品。在冰上预冷1.5 ml微量离心管(每个样品1个)。      


注意:步骤B2-B10必须在4°C下完成。将材料放在冰上或在冷藏室中工作。


2.从-80°C冰箱中取出50 ml装有滤膜和细胞的锥形管,在冰上融化10分钟。      


3.用900 µl冷冻无菌dH 2 O将细胞从滤膜上洗下来,并以最高速度涡旋30 s。将悬浮液转移至冷却的1.5 ml微量离心管中,并置于冰上。      


4.第二次用额外的900μl冷冻无菌dH 2 O洗涤过滤器,并以最高速度涡旋30 s。将悬浮液转移到相同的1.5 ml冷冻离心管中,置于冰上。      


5.对每个样品使用新的试管,对所有样品重复步骤B3和B4。      


6.在标准台式微量离心机中以最大速度(≥17,100 xg )离心30 s,并弃去上清液。      


7.加入600 µl准备好的RLT + 1%BME溶液,并以最大速度涡旋重悬。      


8.将600 µl细胞悬液转移至装有氧化锆珠和苯酚:氯仿:异戊醇的冷螺帽管中。      


9.使用BioSpec MiniBeadBeater-16 607型在4°C下拍打3分钟。      


10.在标准台式微量离心机中,在4°C下以最大速度将细胞离心8分钟。   


11.将550 µl水层转移至新的无RNase的1.5 ml微量离心管中,加入550 µl 70%EtOH,颠倒6次进行混合。   


注意:如果没有550 µl水溶液,则测量并转移到新管中,并加入等体积的70%乙醇。


12.转移到700微升样品,以RNeasy旋转柱并离心15秒的在≥ 8000 ×g离心。丢弃流通。用剩余的样品重新装填色谱柱,然后再次旋转以丢弃流通液。   


注意:Qiagen RNeasy迷你试剂盒规程中提供了步骤B12-B17中提到的所有耗材,用于从酵母中纯化总RNA。该协议的步骤B12-B16与Qiagen RNeasy Mini Kit协议从酵母中纯化总RNA的步骤2-5相同。


13.将700 µl RW1缓冲液添加到色谱柱中,并在≥8,000 xg的转速下旋转15 s 。丢弃流通。   


14.将500 µl缓冲液RPE(添加了乙醇)添加到色谱柱中,并以≥8,000 xg的转速旋转15 s 。丢弃流通。   


15.重复步骤B14。   


16.将旋转柱转移到新的收集管中,并以≥8,000 xg旋转1分钟。丢弃流通。   


17.将旋转柱转移至RN的无硒1.5 ml微量离心管(来自试剂盒)。向柱膜中加入40 µl无RNase的水,并以≥8,000 xg离心1分钟。   


18.取40μL从步骤B17和洗脱液在≥再次重新应用到离心柱膜和旋转1分钟8000 ×g离心。   


注意:这不是严格必要的,但是会增加总浓度。


19.立即在-80°C下冷冻样品进行保存。   


20.为了定量RNA和评估纯度,制备1:总RNA和测量OD 500稀释260使用的Eppendorf BioPhotometer生物分光光度计和Uvette 220-1 ,600个纳米比色皿。   


 注意事项:


平均产量通常超过2 µg / µl,最高约为6 µg / µl。260/280和260/230值通常分别在2.0-2.2和2.5-2.5之间。
根据可用的设备,可能不需要稀释和使用Uvettes 。
 


笔记


 


1 编写此方案可容纳6个样品(2个菌株,每个菌株3个重复样品),但是可以扩展,因为它可以在收获的细胞被冷冻的同时延长数天。它也可以适应RPMI + 10%FBS以外的任何介质条件。但是,如果添加更多的复制品和菌株,建议如果可能的话,提前制作大批培养基,以消除批次对菌株转录谱的影响。例如,在这里我们要求为6个样品提供200 ml的RPMI + 10%FBS,这需要额外的50ml。如果需要60个样品,则应大批量配制1.5 L RPMI + 10%FBS,然后分装成较小的单元,在几天内使用。
2 该协议不包括DNase处理。这意味着最终产品可能会有一些DNA污染。Qiagen RNeasy Mini Kit旋转柱通过设计可防止大多数DNA残留,但可能会残留残余DNA。如果必须避免DNA污染用于下游应用,则可以在S teps B 12和B 13之间的旋转柱上或在最终洗脱后进行DNase处理,有关此操作的规程可以在Qiagen RNeasy Mini Kit规程中找到。
 


菜谱


 


1 YPD液体生长培养基
用500 ml dH 2 O溶解20 g葡萄糖,20 g蛋白ept和10 g酵母提取物
用dH 2 O将体积升至1 L
高压灭菌器消毒
2 10 N氢氧化钠
将40 g NaOH溶解在50 ml dH 2 O中
用dH 2 O将体积提高到100 ml
3 RPMI + 10%胎牛血清
a 将3.28 g RPMI1640粉末溶于180 ml dH 2 O
b 使用10 N NaOH(大约180 µl)将pH调整至7.4
c 通过真空过滤灭菌RPMI溶液
d 在37°C水浴中解冻胎儿牛血清精华(FBS)
注意:FBS制造商建议避免重复的冻融循环,因此要进行相应的计划。


e 轻轻摇动以重悬所有固体
f 将20毫升融化的FBS加入200毫升RPMI溶液中,并轻轻摇晃以免起泡沫
g 储存在4°C下长达一个月
 


致谢


 


NIH赠款1R21AI144878-01和1R01AI146103 (PI:Mitchell)支持在此实验室中对白色念珠菌RNA的研究。该协议从1988年以来在Mitchell实验室中使用的协议演变而来。              


 


利益争夺


 


作者声明不存在利益冲突。


 


参考文献


 


1 Rodríguez,A.,Vaneechoutte ,M.(2019年)。比较不同细胞裂解方法和商业方法从RNAlater中存储的白色念珠菌提取RNA的效率。BMC Microbiol 19(94)。
2 铃木T.和岩桥Y.(2013年)。使用消化方法制备啤酒酵母的RNA可能会产生误导性的结果。应用生物化学生物技术。169(5):1620-1632。
3 Dikicioglu ,D.,Karabekmez ,E.,Rash,B.,Pir ,P.,Kirdar ,B.和Oliver,SG(2011)。酵母如何根据不同的营养素冲动重新编程其转录谱。BMC Syst Biol 5:148。
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引用:Cravener, M. V. and Mitchell, A. P. (2020). Candida albicans Culture, Cell Harvesting, and Total RNA Extraction. Bio-protocol 10(21): e3803. DOI: 10.21769/BioProtoc.3803.
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