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Aug 2013

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siRNA Screening for Genes Involved in HSV-1 Replication
对参与HSV-1病毒复制的基因进行siRNA筛选   

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Abstract

Small interfering RNAs (siRNAs) are small (typically 18-24 nucleotides) RNA molecules capable of silencing gene expression post-transcriptionally and as such, they provide a simple method by which the role of individual genes in complex cellular systems can be easily assessed. As siRNAs are easy to use experimentally, and can be designed to target any gene (including pathogens), their use is perfectly suited to and easily adapted to high-throughput genome-wide screening methodologies and a range of phenotypic assays. Here we describe the use of a large siRNA library (>8,000 genes targeted individually) to screen for and identify host factors functionally involved in the replication of a human herpesvirus (Herpes simplex virus type 1; HSV-1) (Griffiths et al., 2013; Griffiths, 2013).

Materials and Reagents

  1. Hela cells (ATCC, catalog number: CCL-2 )
  2. siRNA library (e.g. human siGENOME SMARTpool Druggable Genome siRNA library 0.5 nmol) (Thermo Fisher Scientific, catalog number: G-004605-05 )
  3. 5x siRNA buffer (Thermo Fisher Scientific, catalog number: B-002000-UB-100 )
  4. RISC-free (RSCF) control siRNA (20 nmol) (Thermo Fisher Scientific, catalog number: D-001220-01-20 )
  5. Scrambled, non-targeting control siRNA (20 nmol, human) (Thermo Fisher Scientific, catalog number: D-001206-13-20 )
  6. Assay-specific control siRNAs
  7. Fetal bovine serum (FBS) (LabTech, catalog number: FCS-SA-10454 )
  8. Penicillin:streptomycin (5,000 units/ml each) (Lonza, catalog number: DE17-603E )
  9. 1x trypsin-EDTA liquid (0.05% Trypsin, 0.53 mM EDTA-4Na) (Life Technologies, catalog number: 25300096 )
  10. Phosphate buffered saline without magnesium or calcium (Lonza, catalog number: 17-516F )
  11. DMEM 4.5 g glucose/litre with L-glutamine and sodium pyruvate (Lonza, catalog number: BE12-604F )
  12. 1x DMEM/F12 1:1 (15 mM HEPES + L-glutamine, no phenol red) (Life Technologies, catalog number: 11039021 )
  13. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, catalog number: HYC-001-181W )
  14. Dharmafect 1 siRNA transfection reagent (Thermo Fisher Scientific, catalog number: T-2001 )
  15. Sterile DNase and RNase-free distilled water (Life Technologies, catalog number: 10977-049 )
  16. Ethanol (diluted in sterile RNase and DNase-free water)
  17. CellTiter-Blue® cell viability reagent (Promega Corporation, catalog number: G8081 )
  18. HSV-1-eGFP reporter virus (Arthur et al., 2001)
  19. Growth medium (see Recipes)
  20. Transfection medium (see Recipes)

Equipment

  1. Pipette tips for robotic liquid handler (Axygen Zymark 200 µl sterile tips) (VWR International, catalog number: AXYGZT-200-L-R-S )
  2. Polystyrene plate covers for 96-well plates (Thermo Fisher Scientific, catalog number: AB0752 )
  3. Aluminium heat-sealing film for storage microplates (Thermo Fisher Scientific, catalog number: AB-0559 )
  4. Adhesive plate seals (Starlab, catalog number: E2796-9793 )
  5. 384-well storage plates (Thermo Fisher Scientific, catalog number: AB-0781 )
  6. Black 384-well clear flat-bottomed, sterile, tissue culture-treated plates (VWR International, catalog number: 734-1640 )
  7. 75 cm2 filter cap tissue culture flasks (Sigma-Aldrich, catalog number: C7106 )
  8. 175 cm2 filter cap tissue culture flasks (Sigma-Aldrich, catalog number: C7481 )
  9. 50 ml centrifuge tube (Corning, product number: 430290 )
  10. Disposable hemocytometer (KOVA Glasstic slide 10 with counting grid) (HYCOR Biomedical, catalog number: 87144E )
  11. Class II Microbiological safety cabinet
  12. Humidified cell culture incubator (37 °C, 5% CO2)
  13. Pipette aid (Alpha laboratories, catalog number: 4-131-201-E )
  14. Centrifuge (e.g. Eppendorf, catalog number: 5811-000.010 )
  15. Light microscope
  16. 8-channel multichannel pipette (2-10 μl volume) (Thermo Fisher Scientific, catalog number: 4661000 )
  17. Multidrop 384 reagent dispenser (Thermo Fisher Scientific, catalog number: 5840150 )
  18. Multidrop 384 standard tube dispensing cassette (Thermo Fisher Scientific, catalog number: 24072670 )
  19. RapidPlate 384 robotic liquid handler (or equivalent) (discontinued) (QIAGEN)
  20. Microplate heat sealer (e.g. ALPS 50 V Microplate heat sealer) (Thermo Fisher Scientific, catalog number: AB-1443 )
  21. Gas-permeable adhesive microplate seals (Thermo Fisher Scientific, catalog number: AB-0718 )
  22. Barcode printer (e.g. Brady® LABXPERT™ printer) (Sigma-Aldrich, catalog number: Z664588 )
  23. Brady® LABXPERT™ printer labels (Thermo Fisher Scientific, catalog number: 12879858 )
  24. Fluorescent plate reader (e.g. POLARstar OPTIMA, BMG LABTECH)
  25. Stacker for plate reader (BMG LABTECH)

Software

  1. Microsoft Excel

Procedure

  1. Remove the lyophilised siRNA library (76 x 96-well plates) from storage at -80 °C and equilibrate to room temperature.
  2. Pellet well contents by centrifuging for 10 min at 1,000 rpm and replace plate seals with clean plastic lids.
  3. Clean a Multidrop 384 dispensing cassette with 3 x 25 ml 70% ethanol and rinse with 3 x 50 ml sterile DNase and RNase-free water.
  4. Prepare a sufficient volume of 1x siRNA buffer by diluting 5x siRNA buffer in sterile DNase and RNase-free water.
  5. Resuspend the 0.5 nmol siRNA library to 3 µM by dispensing 165 µl 1x siRNA buffer to columns 3-12 of the siRNA library plates (96-well plates) (see Note 1; Figure 1A).
  6. Using a label maker, print text or barcode labels for 384-well Master Plates (see Note 2).
  7. Adhere labels to 384-well storage plates (for a library of 76 x 96-well plates you will need 19 x 384-well storage plates and labels). These will be the 384-well Master Plates.
  8. Clean a dispensing cassette for the Multidrop 384, as above, and dispense 110 µl 1x siRNA buffer into columns 1 and 2 of the 384-well Master plate (see Note 3; Figure 1B).


    Figure 1. 96 and 384-well plate preparation

  9. Dilute the assay-specific siRNA controls to 300 nM in 1x siRNA buffer (see Note 4).
  10. Manually transfer 110 µl of each siRNA control, in horizontal duplicates, to columns 3 and 4 in every 384-well Master Plate i.e. transfer RSCF siRNA to wells A3 and A4; transfer non-targeting siRNA to wells B3 and B4 etc. (Figure 1C).
  11. Dispense 100 µl 1x siRNA buffer to columns 5-24 in each 384-well master plate using a Multidrop 384.
  12. Using a robotic liquid handler (such as the QIAGEN RapidPlate 384), with an individual box of Zymark robotic tips for each siRNA 96-well Library Plate, transfer 11 µl from the siRNA library plates (3 µM siRNA) to the 384-well Master plate. Each 384-well plate takes 4 x 96-well siRNA Library plates (Figure 1D). These plates can be stored at -80 °C until needed.
  13. Use a microplate heat sealer and aluminium heat seals to seal the siRNA library plates before transferring to a -80 °C for long-term storage.
  14. Print appropriate barcode or text labels for the 384-well assay plates containing relevant information (replicate number, assay type etc.) (see Note 5).
  15. Stick labels to black, tissue-culture treated, flat, clear-bottomed 384-well plates.
  16. If frozen, thaw the 384-well Master Plates and briefly centrifuge for 1 min at 1,000 rpm before transferring 10 µl to three replicate assay plates using a robotic liquid handler and an individual box of Zymark robotic tips for each quadrant of the 384-well plate.
  17. Cover prepared assay plates with an adhesive plate seal, place in sealable bags and stored at -80 °C for up to 2 weeks before use (see Note 6).
  18. Calculate the number of low passage Hela cells in a confluent T175 flask, and the total number of cells required for your assay. Based on these numbers determine the number of 50% confluent T175 flasks required to provide the total number of cells for your assay.
  19. Several days before your assay, prepare Hela cells such that there will be sufficient 100% confluent T175 flasks the day before the siRNA transfection.
  20. The day before transfection, remove medium from the confluent T715 flasks of Hela cells, and rinse cells with warmed sterile phosphate buffered saline.
  21. Discard wash, and dislodge cells by incubating for around 5 min at 37 °C in 5 ml pre-warmed trypsin.
  22. Inactivate trypsin by adding 10 ml growth medium and transfer the pre-determined volume of cells to sufficient T175 flasks such that they will be 50% confluent the following day (see Note 7).
  23. The next day, thaw the replicate assay plates and centrifuge for 5 min at 1,000 rpm. Remove the plate seals and cover with lids.
  24. Dilute the Dharmafect 1 (DF1) transfection reagent to 0.6% in the appropriate volume of HBSS and add 10 µl to each well of the 384-well assay plates using a dispensing cassette for the Multidrop 384.
  25. Centrifuge the assay plates for 1 min at 1,000 rpm and incubate at room temperature for 20 min for transfection complexes to form.
  26. Remove growth medium from the Hela cells, rinse in PBS and dislodge cells by incubating for ~5 min at 37 °C in 5 ml trypsin.
  27. Inactivate the trypsin by adding 10 ml of transfection medium.
  28. Mix each flask well and pool together all cells before mixing well and removing 5 x 12 μl aliquots of cell suspension for counting in a disposable hemocytometer.
  29. Calculate the average cell density and dilute to 7.5 x 104 cells/ml.
  30. After the 20 min incubation time, transfer 40 µl cells to every well in the assay plates and incubate plate in a humidified incubator at 37 °C for 48 h to ensure gene depletion prior to investigating your assay output.
  31. After 48 h, determine the effect of gene depletion on cell viability.
  32. Thaw the CellTitre Blue reagent (or equivalent) and add 5 µl per well of each Cell Viability replicate plate using the Multidrop 384.
  33. Return the plates to the 37 °C incubator for 2 h before measuring the fluorescence signal (485 nm) of each plate using a POLARstar OPTIMA plate reader (or equivalent). The effect of siRNA depletion on cell viability is determined by comparing fluorescence to the average fluorescence of mock-transfected cells (columns 1 and 2) (see Note 8).
  34. To infect the duplicate assay plates with HSV-1-eGFP, thaw theHSV-1-eGFP virus stock in a 37 °C water bath until just thawed, before diluting to a multiplicity of infection (MOI) of 0.5 in phenol red-free DMEM: F12/15 mM HEPES/L-glutamine/5% FCS/pen-strep (see Note 9).
  35. Remove media from all plates by inverting and shaking over a suitable liquid waste container.
  36. Add 10 µl phenol red-free growth medium to columns 1 and 2 of every plate manually using a low volume (2-10 µl) 8-channel multichannel pipette (see Note 10).
  37. Add 10 µl diluted virus to the remaining wells (columns 3-24) using a Multidrop 384 and centrifuge for 2 min at 1,000 rpm to remove air bubbles and ensure the cell monolayer is covered by the small volume of inoculum.
  38. Transfer infection assay plates to the 37 °C incubator for 1 h to allow virus to adsorb before adding 50 µl phenol red-free growth medium to every well using the Multidrop 384.
  39. Cover the plates with a gas-permeable adhesive seal, stack and cover the top plate with a plastic lid before returning all assay plates to the incubator for 20 h.
  40. Place assay plates in the stacker equipment of a fluorescence plate reader and monitor virus replication as a measure of GFP fluorescence in a fluorescent plate reader every 3-4 h until replication plateaus (see Note 11).
  41. Once the virus replication cycle has completed, calculate the replication slope over the linear phase of growth for each well and determine the effect of gene depletion on virus replication by comparing the replication slope to the average replication slope of control wells (mock, RSCF and non-targeting siRNA-transfected) on a per plate basis. The LINEST equation in Microsfot Excel can be used to calculate replication slopes. This formula utilises the ‘known x’ values (the hours post-infection) and the ‘known y’ values (the relative fluorescence at each time post-infection). Firstly, generate a table with the ‘Hours post-infection’ and the relative fluorescence of your samples (Figure 1A). Plot these data and identify the linear phase of replication (Figure 1B; red line indicates linear phase). Use the linest formula to calculate the replication slope, and normalise the slope of each condition to that of your control 9in this example, normalise to ‘Mock’ transfected cells; Figure 1C-D).



    Figure 2. Calculation of replication slopes

Notes

  1. Library plates are diluted 1: 10 to 300 nM when generating the 384-well Master Plates, and diluted a further 1: 6 during the assay. This gives a final siRNA concentration of 50 nM. If a higher/lower siRNA concentration is preferred, adjust the resuspension volumes accordingly. columns 1 and 2 of the 96-well siRNA Library plates are empty to enable the user to include assay-specific controls.
  2. As siRNAs are temperature sensitive, we recommend the generation of a 384-well master plate which contains sufficient siRNA to complete three full screens in triplicates. siRNA can be dispensed into replicate assay plates at the same time to ensure an equal number of freeze-thaw cycles of assay plates and reproducible assay results.
  3. The addition of siRNA buffer alone to columns 1 and 2 of each 384-well master plate will result in untransfected control cells. For our virus replication assay, these will remain uninfected.
  4. Be sure to include mock-transfected cells (transfection reagent alone), siRNA controls (such as RISC-free and a scrambled, non-targeting siRNA) and assay-specific siRNAs which display a known range of phenotypes (strong positive effect, moderate effect, strong negative effect).
  5. For a virus replication assay we recommend carrying out each assay in triplicate. One replicate should be used for a cell viability assay (as virus will not replicate if siRNA gene depletion has a cytotoxic effect) and the virus replication assay is done in duplicates.
  6. Whilst diluted siRNA will be stable for some time at -80 °C the risk of evaporation of such a small volume is high. We would recommend storing assay plates for no longer than 2 weeks before use.
  7. siRNA transfection and virus replication is optimal when cells were sub-confluent (~50%) and freshly passaged prior to use.
  8. The Cell Titre Blue reagent is metabolised to produce a fluorescent signal. The cell viability is therefore demonstrated by the rate of metabolism of the cells. The effect of gene depletion on cell viability is determined by comparing the fluorescence of siRNA-transfected wells to the average fluorescence of control, non-transfected wells (columns 1 and 2) on a per plate basis to avoid inter-plate variations. Normalising the fluorescence in this way enables identification of cytotoxic siRNAs as well as those increase cell proliferation. As per manufacturer’s instructions, a fluorescence value of <70% of the control cells is considered cytotoxic. Cytotoxic siRNAs are excluded from subsequent data analyses.
  9. It is important to infect cells with a known quantity of virus (multiplicity of infection; MOI) to generate a suitable growth curve. We find a multiplicity of infection (MOI) of 0.5 [0.5 virus plaque forming units (PFU) per cell] gives an ideal kinetic growth curve from 20 to 80 h post-infection. The MOI is dependent upon the cell number and volume used for the infection, so the appropriate dilution factor should be calculated based upon the starting titre of your virus stock. For example, to infect 3 x 103 cells at MOI 0.5 in 10 μl we need to have a virus preparation containing 1.5 x 105 PFU/ml. A starting stock of 6 x 107 PFU/ml should therefore be diluted 1 in 400.
  10. Tissue culture plasticware, cells and growth medium all have some level of fluorescence. When utilising fluorescent reporter genes it is therefore essential to have appropriate controls to provide background fluorescence readings. For this assay, the fluorescence from uninfected cells is used as background.
  11. Replication is monitored over regular intervals to enable a complete growth curve (fluorescence over time) to be plotted. When comparing HSV replication between untreated and treated samples we use the slope of replication over the linear growth phase. It is therefore important to have as many measurements over this phase as possible (with a minimum of 6 for statistical reliability of the slope calculation). A test replication assay with your own equipment will allow the time of linear growth to be established, and assay timings can be adjusted to ensure measurements can be taken over this period.

Recipes

  1. Growth medium
    DMEM
    5% FCS
    1% pen-strep
  2. Transfection medium
    Phenol red-free DMEM: F12
    15 mM HEPES
    L-glutamine
    5 % FCS

Acknowledgments

This protocol has been edited from that described in Griffiths et al. (2013) PLOS Pathogens. The authors are grateful to Prof. Peter Ghazal for provision of the siRNA libraries, and Marie Craigon for technical assistance with library handling, management and establishment of protocols. The authors are grateful to the MRC (G0501453 J.H.), BBSRC (BB/B019621/1 P.G.), Scottish Enterprise (P.G.) and the Wellcome Trust (WT066784 P.G.) for funding.

References

  1. Arthur, J. L., Scarpini, C. G., Connor, V., Lachmann, R. H., Tolkovsky, A. M. and Efstathiou, S. (2001). Herpes simplex virus type 1 promoter activity during latency establishment, maintenance, and reactivation in primary dorsal root neurons in vitro. J Virol 75(8): 3885-3895.
  2. Griffiths, S. J., Koegl, M., Boutell, C., Zenner, H. L., Crump, C. M., Pica, F., Gonzalez, O., Friedel, C. C., Barry, G., Martin, K., Craigon, M. H., Chen, R., Kaza, L. N., Fossum, E., Fazakerley, J. K., Efstathiou, S., Volpi, A., Zimmer, R., Ghazal, P. and Haas, J. (2013). A systematic analysis of host factors reveals a Med23-interferon-lambda regulatory axis against herpes simplex virus type 1 replication. PLoS Pathog 9(8): e1003514.
  3. Griffiths, S. J. (2013). Screening for host proteins with pro- and anti-viral activity using high-throughput RNAi in ‘Virus-Host Interactions’. In: Methods in Molecular Biology. Vol. 1064: 71-90.

简介

小干扰RNA(siRNA)是能够沉默转录后基因表达的小(通常为18-24个核苷酸)RNA分子,因此,它们提供了一种简单的方法,通过其可以容易地评估单个基因在复杂细胞系统中的作用。 由于siRNA易于实验使用,并且可以设计为靶向任何基因(包括病原体),它们的使用完全适合于并且容易地适应于高通量全基因组筛选方法学和一系列表型测定。 这里我们描述了使用大的siRNA文库(> 8,000个基因单独靶向)来筛选和鉴定功能上涉及人疱疹病毒(1型单纯疱疹病毒; HSV-1)(Griffiths)的复制的宿主因子, et al。,2013; Griffiths,2013)。

材料和试剂

  1. Hela细胞(ATCC,目录号:CCL-2)
  2. siRNA文库(例如人siGENOME SMARTpool Druggable Genome siRNA文库0.5nmol)(Thermo Fisher Scientific,目录号:G-004605-05)
  3. 5x siRNA缓冲液(Thermo Fisher Scientific,目录号:B-002000-UB-100)
  4. 无RISC(RSCF)对照siRNA(20nmol)(Thermo Fisher Scientific,目录号:D-001220-01-20)
  5. 加扰的非靶向对照siRNA(20nmol,人)(Thermo Fisher Scientific,目录号:D-001206-13-20)
  6. 测定特异性对照siRNA
  7. 胎牛血清(FBS)(LabTech,目录号:FCS-SA-10454)
  8. 青霉素:链霉素(每种5,000单位/ml)(Lonza,目录号:DE17-603E)
  9. 1x胰蛋白酶-EDTA液体(0.05%胰蛋白酶,0.53mM EDTA-4Na)(Life Technologies,目录号:25300096)
  10. 不含镁或钙的磷酸盐缓冲盐水(Lonza,目录号:17-516F)
  11. DMEM 4.5g葡萄糖/升与L-谷氨酰胺和丙酮酸钠(Lonza,目录号:BE12-604F)
  12. 1×DMEM/F12 1:1(15mM HEPES + L-谷氨酰胺,无酚红)(Life Technologies,目录号:11039021)
  13. 汉克平衡盐溶液(HBSS)(Thermo Fisher Scientific,目录号:HYC-001-181W)
  14. Dharmafect 1siRNA转染试剂(Thermo Fisher Scientific,目录号:T-2001)
  15. 无菌DNase和无RNase蒸馏水(Life Technologies,目录号:10977-049)
  16. 乙醇(稀释于无菌RNase和无DNase的水中)
  17. CellTiter-Blue细胞活力试剂(Promega Corporation,目录号:G8081)
  18. HSV-1-eGFP报道病毒(Arthur等人,2001)
  19. 生长培养基(参见食谱)
  20. 转染培养基(见配方)

设备

  1. 用于机器人液体处理器的移液器吸头(Axygen Zymark200μl无菌吸头)(VWR International,目录号:AXYGZT-200-L-R-S)
  2. 用于96孔板(Thermo Fisher Scientific,目录号:AB0752)的聚苯乙烯板盖
  3. 用于存储微板的铝热封膜(Thermo Fisher Scientific,目录号:AB-0559)
  4. 粘合板密封件(Starlab,目录号:E2796-9793)
  5. 384-孔培养板(Thermo Fisher Scientific,目录号:AB-0781)
  6. 黑色384孔透明平底,无菌,组织培养处理的平板(VWR International,目录号:734-1640)
  7. 75cm 2过滤盖组织培养烧瓶(Sigma-Aldrich,目录号:C7106)
  8. 175cm 2过滤盖组织培养烧瓶(Sigma-Aldrich,目录号:C7481)
  9. 50ml离心管(Corning,产品编号:430290)
  10. 一次性血细胞计数器(KOVA Glasstic slide 10 with counting grid)(HYCOR Biomedical,目录号:87144E)
  11. II类微生物安全柜
  12. 加湿的细胞培养孵育器(37℃,5%CO 2)
  13. 移液器辅助(Alpha实验室,目录号:4-131-201-E)
  14. 离心机(例如:Eppendorf,目录号:5811-000.010)
  15. 光学显微镜
  16. 8通道多通道移液管(2-10μl体积)(Thermo Fisher Scientific,目录号:4661000)
  17. Multidrop 384试剂分配器(Thermo Fisher Scientific,目录号:5840150)
  18. Multidrop 384标准管分配盒(Thermo Fisher Scientific,目录号:24072670)
  19. RapidPlate 384机器人液体处理器(或同等产品)(停产)(QIAGEN)
  20. 微孔板热封机(例如ALPS 50V微板热封机)(Thermo Fisher Scientific,目录号:AB-1443)
  21. 气体可渗透粘合剂微板密封件(Thermo Fisher Scientific,目录号:AB-0718)
  22. 条形码打印机(例如 Brady ® LABXPERT™打印机)(Sigma-Aldrich,目录号:Z664588)
  23. Brady ® LABXPERT™打印机标签(Thermo Fisher Scientific,目录号:12879858)
  24. 荧光板读数器(例如 POLARstar OPTIMA,BMG LABTECH)
  25. 读板器堆垛机(BMG LABTECH)

软件

  1. Microsoft Excel

程序

  1. 在-80℃下从存储中取出冻干的siRNA文库(76×96-孔板),并平衡至室温。
  2. 通过在1,000rpm下离心10分钟来沉淀孔内容物,并用干净的塑料盖替换板密封件
  3. 用3×25ml 70%乙醇清洁Multidrop 384分液盒,并用3×50 ml无菌DNase和无RNase的水冲洗。
  4. 通过在无菌DNase和无RNase的水中稀释5x siRNA缓冲液制备足够体积的1x siRNA缓冲液。
  5. 通过将165μl1×siRNA缓冲液分配到siRNA文库平板(96孔板)的3-12列,将0.5nmol siRNA文库重悬于3μM(参见注释1;图1A)。
  6. 使用标签制造商,为384孔主板打印文本或条形码标签(参见注释2)。
  7. 将标签贴在384孔培养板上(对于76 x 96孔培养板,您需要19 x 384孔培养板和标签)。这些将是384孔主板。
  8. 如上所述清洁Multidrop 384的分液盒,并在384孔Master板的第1和2列中分配110μl1x siRNA缓冲液(见注3;图1B)。
  9. 在1x siRNA缓冲液中稀释测定特异性siRNA对照至300nM(参见注释4)
  10. 在每个384孔Master板中将110μl的每个siRNA对照以水平重复的方式手动转移到3和4号孔中,即将RSCF siRNA转移到孔A3和A4中;将非靶向siRNA转移到孔B3和B4等。(图1C)
  11. 使用Multidrop 384将100μl1x siRNA缓冲液分配到每个384孔主板中的柱5-24
  12. 使用机器人液体处理器(例如QIAGEN RapidPlate 384),每个siRNA 96孔文库板具有单独的Zymark机器人吸头盒,将11μl来自siRNA文库平板(3μMsiRNA)转移至384孔Master盘子。每个384孔板需要4 x 96孔siRNA文库板(图1D)。这些板可以储存在-80℃直到需要
  13. 使用微孔板热封机和铝热封来密封siRNA库板,然后转移至-80°C长期保存。
  14. 为包含相关信息(重复数,测定类型等)的384孔测定板打印适当的条形码或文本标签(见注5)。
  15. 将标签贴到黑色,组织培养处理的,平的,透明底的384孔板上
  16. 如果冷冻,解冻384孔主板,并简单离心1分钟,在1,000 rpm,然后使用机器人液体处理程序和一个单独的盒的Zymark机器人吸头转移10微升到三个复制测定板384孔板的每个象限。
  17. 盖上准备的测定板与粘性板密封,放置在可密封的袋和使用前储存在-80℃长达2周(见注6)。
  18. 计算融合T175烧瓶中低通道Hela细胞的数量,以及您的测定所需的细胞总数。基于这些数量确定提供用于测定的细胞总数所需的50%融合T175烧瓶的数量。
  19. 在测定前几天,制备Hela细胞,使得在siRNA转染前一天有足够的100%汇合的T175培养瓶。
  20. 转染前一天,从汇合的T715细胞的Hela细胞中取出培养基,并用温热的无菌磷酸盐缓冲盐水冲洗细胞。
  21. 弃去洗涤,并通过在37℃下在5ml预热的胰蛋白酶中孵育约5分钟来移出细胞。
  22. 通过加入10ml生长培养基来灭活胰蛋白酶,并将预定体积的细胞转移到足够的T175烧瓶中,使得它们在第二天达到50%汇合(参见注释7)。
  23. 第二天,解冻重复测定板并在1,000rpm离心5分钟。用盖子拆下板密封件和盖子。
  24. 在适当体积的HBSS中将Dharmafect 1(DF1)转染试剂稀释至0.6%,并使用Multidrop 384的分配盒将10μl加入384孔测定板的每个孔中。
  25. 将测定板在1,000rpm离心1分钟,并在室温下孵育20分钟,以形成转染复合物。
  26. 从Hela细胞中移除生长培养基,在PBS中冲洗,并通过在37℃下在5ml胰蛋白酶中孵育〜5分钟来移出细胞。
  27. 加入10ml转染培养基使胰蛋白酶失活。
  28. 将每个烧瓶充分混合,将所有细胞混合在一起,并混合均匀,取出5×12μl等分的细胞悬液,在一次性血细胞计数器中计数。
  29. 计算平均细胞密度并稀释至7.5×10 4细胞/ml
  30. 在20分钟孵育时间后,将40微升细胞转移到测定板中的每个孔中,并在37℃下在湿润的培养箱中培养板48小时,以确保在调查测定输出之前基因消耗。
  31. 48小时后,确定基因耗尽对细胞活力的影响
  32. 解冻CellTitre蓝色试剂(或同等物),并使用Multidrop 384每孔加入5μl每个细胞活力复制板。
  33. 将板放回37℃培养箱中2小时,然后使用POLARstar OPTIMA板读数器(或等效物)测量每个板的荧光信号(485nm)。通过比较荧光与模拟转染细胞的平均荧光(第1列和第2列)(参见注释8)来确定siRNA耗尽对细胞活力的影响。
  34. 为了用HSV-1-eGFP感染重复测定平板,在37℃水浴中解冻HSV-1-eGFP病毒储液,直到刚解冻,然后在不含酚红的DMEM中稀释至0.5的感染复数(MOI) :F12/15mM HEPES/L-谷氨酰胺/5%FCS/pen-strep(见注9)。
  35. 通过翻转并摇动合适的液体废物容器,从所有板中取出介质。
  36. 使用低体积(2-10μl)8通道多通道移液器(见注10)手动添加10μl无酚红生长培养基到每个板的第1列和第2列。
  37. 使用Multidrop 384在剩余的孔(3-24列)中加入10μl稀释的病毒,并在1,000 rpm离心2分钟以除去气泡,确保细胞单层被小体积的接种物覆盖。
  38. 转移感染测试平板到37°C孵育器1小时,以允许病毒吸附,然后使用Multidrop 384添加50μl无酚红生长培养基到每个井。
  39. 用气体可渗透的粘性密封盖住板,堆叠并用塑料盖覆盖顶板,然后将所有测定板返回培养箱20小时。
  40. 将测定板放在荧光板读数器的堆叠器设备中,并监测病毒复制,作为每3-4小时荧光读板器中GFP荧光的测量,直到复制平台(参见注释11)。
  41. 一旦病毒复制周期完成,计算每个孔的生长的线性期的复制斜率,并通过将复制斜率与对照孔的平均复制斜率比较来确定基因缺失对病毒复制的影响(模拟,RSCF和非 - 靶向siRNA转染)。 Microsfot Excel中的 LINEST 方程式可用于计算复制斜率。该公式利用"已知的x"值(感染后的小时)和"已知的y"值(感染后的每个时间的相对荧光)。首先,生成一个具有"感染后小时"和样品的相对荧光的表格(图1A)。绘制这些数据并确定复制的线性相(图1B;红线表示线性相)。使用 linest 公式计算复制斜率,并将每个条件的斜率归一化为对照9的斜率,在此示例中,归一化为'Mock'转染的细胞;图1C-D)。


    图1. 96和384孔板制备


    图2.复制斜率计算

笔记

  1. 当产生384-孔主板时,文库板以1:10-300nM稀释,并在测定期间进一步稀释1:6。这得到50nM的最终siRNA浓度。如果优选较高/较低的siRNA浓度,则相应地调整重悬浮体积。 96孔siRNA文库板的第1和2列是空的,以使用户能够包括测定特异性对照
  2. 由于siRNA是温度敏感的,我们建议生成384孔主板,其含有足够的siRNA以一式三份完成三个全屏。 siRNA可以同时分配到重复测定平板中以确保测定平板的相同数目的冻融循环和可重现的测定结果。
  3. 将siRNA缓冲液单独添加到每个384孔主板的柱1和2中将导致未转染的对照细胞。对于我们的病毒复制测定,这些将保持未感染
  4. 确保包括模拟转染的细胞(单独的转染试剂),siRNA对照(例如无RISC和乱序,非靶向siRNA)和测定特异性siRNA,其显示已知范围的表型(强阳性效应,中度效应,强负面影响)。
  5. 对于病毒复制测定,我们建议进行每个测定一式三份。一个重复应该用于细胞生存力测定(如果siRNA基因缺失具有细胞毒性效应,则病毒不会复制),并且病毒复制测定重复进行。
  6. 虽然稀释的siRNA在-80℃下将稳定一段时间,但是这样小体积的蒸发的风险高。我们建议在使用前保存测定板不超过2周。
  7. siRNA转染和病毒复制是最佳的,当细胞汇合(〜50%)和使用前新传代。
  8. 细胞滴定蓝试剂被代谢以产生荧光信号。因此,细胞活力通过细胞的代谢速率来证明。通过将siRNA转染的孔的荧光与对照,非转染孔(柱1和2)的平均荧光在每板的基础上进行比较以避免板间变化,来确定基因损耗对细胞活力的影响。以这种方式标准化荧光使得能够鉴定细胞毒性siRNA以及那些增加细胞增殖。根据制造商的说明书,<70%的对照细胞的荧光值被认为是细胞毒性的。细胞毒性siRNA从随后的数据分析中排除
  9. 用已知量的病毒(感染复数; MOI)感染细胞以产生合适的生长曲线是重要的。我们发现0.5 [0.5个病毒噬斑形成单位(PFU)/细胞]的感染复数(MOI)在感染后20至80小时得到理想的动力学生长曲线。 MOI取决于用于感染的细胞数量和体积,因此应基于病毒原液的起始滴度来计算适当的稀释因子。例如,为了以10μl的MOI 0.5感染3×10 3个细胞,我们需要具有含有1.5×10 5 PFU/ml的病毒制备物。因此,起始原料为6×10 7 PFU/ml时,应稀释为400.
  10. 组织培养塑料制品,细胞和生长培养基都具有一定程度的荧光。当使用荧光报告基因时,因此必须具有适当的对照以提供背景荧光读数。对于该测定,将来自未感染细胞的荧光用作背景
  11. 在规则的时间间隔上监测复制以使得能够绘制完整的生长曲线(随时间的荧光)。当比较未处理和处理的样品之间的HSV复制时,我们使用在线性生长期的复制的斜率。因此,重要的是在该相位上尽可能多地进行测量(对于斜率计算的统计可靠性,具有最小值6)。使用您自己的设备进行的测试复制测定将允许建立线性生长的时间,并且可以调整测定时间以确保在这段时间内进行测量。

食谱

  1. 生长培养基
    DMEM
    5%FCS
    1%pen-strep
  2. 转染培养基
    酚红无DMEM:F12
    15 mM HEPES
    L-谷氨酰胺 5%FCS

致谢

该方案是从Griffiths等人(2013)PLOS Pathogens中描述的。 作者感谢Peter Ghazal教授提供siRNA文库,Marie Craigon为图书馆处理,管理和建立方案提供技术援助。 作者感谢MRC(G0501453 J.H.),BBSRC(BB/B019621/1 P.G.),Scottish Enterprise(P.G.)和Wellcome Trust(WT066784 P.G.)的资助。

参考文献

  1. Arthur,J.L.,Scarpini,C.G.,Connor,V.,Lachmann,R.H.,Tolkovsky,A.M.and Efstathiou,S。(2001)。 单纯疱疹病毒1型启动子活性,在初级背根神经元的潜伏期建立,维持和再活化期间< em> in vitro 。 J Virol 75(8):3885-3895。
  2. Griffiths,SJ,Koegl,M.,Boutell,C.,Zenner,HL,Crump,CM,Pica,F.,Gonzalez,O.,Friedel,CC,Barry,G.,Martin,K.,Craigon, Chen,R.,Kaza,LN,Fossum,E.,Fazakerley,JK,Efstathiou,S.,Volpi,A.,Zimmer,R.,Ghazal,P.and Haas,J.(2013)。 对宿主因子的系统分析揭示了针对单纯疱疹病毒1型的Med23-干扰素λ调节轴复制。 PLoS Pathog 9(8):e1003514。
  3. Griffiths,S.J。(2013)。使用高通量RNAi在'病毒 - 宿主相互作用'中筛选具有前和抗病毒活性的宿主蛋白。 In: Methods in Molecular Biology。 1064:71-90。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Griffiths, S. J. and Haas, J. (2014). siRNA Screening for Genes Involved in HSV-1 Replication. Bio-protocol 4(16): e1209. DOI: 10.21769/BioProtoc.1209.
  2. Griffiths, S. J., Koegl, M., Boutell, C., Zenner, H. L., Crump, C. M., Pica, F., Gonzalez, O., Friedel, C. C., Barry, G., Martin, K., Craigon, M. H., Chen, R., Kaza, L. N., Fossum, E., Fazakerley, J. K., Efstathiou, S., Volpi, A., Zimmer, R., Ghazal, P. and Haas, J. (2013). A systematic analysis of host factors reveals a Med23-interferon-lambda regulatory axis against herpes simplex virus type 1 replication. PLoS Pathog 9(8): e1003514.
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