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Jan 2018
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Viral Double-Stranded RNA Detection by DNase I and Nuclease S1 digestions in Leishmania parasites
在利什曼原虫中利用DNase I和 Nuclease S1酶解进行病毒双链RNA鉴定   

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Abstract

Many RNA viruses are found in protozoan parasites. They can be responsible for more serious pathology or treatment failure. For the detection of viral double-stranded RNA (dsRNA), sequence-dependent and -independent methods are available, such as quantitative real-time PCR and immunofluorescence, dot blot, ELISA or sequencing. The technique presented here is sequence-independent and is well detailed in the following protocol, taking the example of Leishmania RNA virus (LRV) in Leishmania guyanensis (Lgy) species. To summarise, the protocol is divided into four major steps: RNA extraction from the parasites, RNA purification, enzymatic digestions with DNase I and Nuclease S1, and visualization by gel electrophoresis. This method can be used to detect other viral dsRNA in other parasites. It provides an additional tool, complementary to other techniques previously cited and it is easy and quite fast to achieve.

Keywords: dsRNA (dsRNA), Virus (病毒), Parasite (寄生虫), LRV (Leishmania RNA virus) (利什曼原虫RNA病毒), Detection (检测), DNase I (DNase I), Nuclease S1 (Nuclease S1)

Background

The wide diversity of RNA viruses present in protozoan parasites has been well documented (Wang and Wang, 1991; Ghosh et al., 2012; Zangger et al., 2014; Lye et al., 2016; Akopyants et al., 2016; Fernandez-Presas et al., 2017; Grybchuk et al., 2018). Moreover, these viruses have been described as potential virulence factors (Fichorova et al., 2013; El-Gayar et al., 2016; Rath et al., 2019). Of particular note, the presence of the endosymbiont Leishmania RNA virus (LRV), a Totiviridae double-stranded RNA (dsRNA) virus, in Leishmania guyanensis (Lgy) exacerbates leishmaniasis disease (Ives et al., 2011; Rossi et al., 2017), favors metastasis by inducing interleukin-17 (Hartley et al., 2016) and also increases the risk of treatment failure (Adaui et al., 2016; Bourreau et al., 2016; Vieira-Gonçalves et al., 2019). This shows the importance of viral dsRNA detection in parasites. The technique presented here is sequence-independent, in comparison to quantitative real-time PCR, so it can be applied widely to RNA viruses. This dsRNA detection protocol can also, and is recommended, to be used to confirm (or be confirmed with) other methods of detection that are sequence-independent or -dependent, as for example dot blot or PCR (Zangger et al., 2013). The technique is presented here with LRV, however its application is possible for other RNA viruses and in other parasites (Grybchuk et al., 2018).

Materials and Reagents

  1. Eco Nitrile PF 250 Gloves (ecoSHIELDTM)
  2. Filter tips Low retention 1,000 µl, 200 µl, 20 µl (ClearLine, catalog numbers: 713118 , 713117 , 713115 , respectively)
  3. 10 µl Extended Length Filter Tip (Neptune Scientific, catalog number: BT10XL )
  4. Tissue culture flask 25 version “Vent” (TPP®, catalog number: 90025 )
  5. Vacuum Filtration 500 “rapid”-Filtermax, PES membrane 0.22 μm pore size (TPP®, catalog number: 99500 )
  6. Microcentrifuge 1.5 ml tubes (Corning, Axygen®, catalog number: MCT-175-C )
  7. Polypropylene conical 50 ml centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050 )
  8. 2 ml serological pipettes (FALCON®, catalog number: 357507 )
  9. 5 ml serological pipettes (SARSTEDT, catalog number: 86.1253.001 )
  10. 10 ml serological pipettes (SARSTEDT, catalog number: 86.1254.001 )
  11. 25 ml serological pipettes (SARSTEDT, catalog number: 86.1685.001 )
  12. Dulbecco's Phosphate-Buffered Saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14040091 ) stored at 4 °C
  13. Schneider’s Drosophila Medium w: L-Glutamine and 0.40 g/L NaHCO3 (PANTM BIOTECH, catalog number: P04-91500 ) stored at 4 °C
  14. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 ), sent at -20 °C, then heat-inactivated (see Recipes from Bio-protocol by Reverte and Fasel, 2019), aliquoted to 50 ml tubes and stored at -20 °C
  15. HEPES Buffer 1 M (BioConcept, catalog number: 5-31F00-H ) stored at 4 °C
  16. Penicillin-Streptomycin (P/S) solution (10,000 IU/ml P and 10 mg/ml S) (Bioconcept, catalog number: 4-01F00-H ), sent at -20 °C, then stored at 4 °C for short-term storage
  17. Hemin BioXtra, from Porcine (Sigma-Aldrich, catalog number: 51280 ) stored at 4 °C
  18. Folic acid (FlukaTM, catalog number: 47620 ) stored at 4 °C
  19. 6-Biopterin (Sigma-Aldrich, catalog number: B2517 ) stored at -20 °C
  20. TRI® reagent (Molecular Research Center, MRC, catalog number: TR118 , 500 ml) stored at 4 °C
  21. Chloroform (Thermo Fisher Scientific, Fisher Chemical, catalog number: C/4960/15 ) stored at room temperature
  22. Isopropanol (Thermo Fisher Scientific, Fisher Chemical, catalog number: P/7490/17 ) stored at room temperature
  23. Ethanol absolute (Thermo Fisher Scientific, Fisher Chemical, catalog number: E/0650DF/17 ) stored at room temperature
  24. UltraPureTM DNase/RNase-free Distilled Water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977-035 ), shipped at room temperature, stored at 4 °C before opening, aliquoted to 1.5 ml tubes and stored at -20 °C
  25. DNase I, RNase-free 10 U/µl with an activity of 0.2 U/µg on calf thymus DNA (Roche, catalog number: 10 776 785 001 ) stored at -20 °C
  26. Nuclease S1 (#E576) (Promega, catalog number: M5761 ) stored at -20 °C
    Note: Pay attention to the fact that the concentration is specific to each tube and is indicated on the tube by the manufacturer.
  27. Buffer S1 (#E578) (Promega, catalog number: M577A ) stored at -20 °C
    Note: The old reference was at 7.4x, but now Nuclease S1 Reaction Buffer is provided at 10x.
  28. 1 kb DNA Ladder 500 µg/ml (NEB, catalog number: N3232S ) stored at 4 °C
  29. 6x Gel Loading Dye (Purple), no SDS (NEB, catalog number: B7025 ) stored at 4 °C; supplied with the 1 kb DNA Ladder
  30. Standard Agarose–Type LE for routine gel electrophoresis (BioConcept Ltd, Amimed, catalog number: 7-01P02-R ) stored at room temperature
  31. Tris(hydroxymethyl)aminomethane, Molecular biology (Biosolve, catalog number: 20092391 ) stored at room temperature
  32. Boric Acid (Crystalline Powder/Electrophoresis grade) (Fisher BioReagents, catalog number: BP168-1 ) stored at room temperature
  33. EDTA, Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate (Crystalline Powder/Electrophoresis grade) (Fisher BioReagents, catalog number: BP120-1 ) stored at room temperature
  34. SDS, Sodium Dodecylsulfate, Solution 20% pure (AppliChem PanReac, ITW Reagents, catalog number: A3942,1000 ) stored at room temperature
  35. Ultra-pure water, type Ultra ClearTM (BLANC-LABO) stored at room temperature
  36. RNase AWAYTM (Molecular BioProductsTM, MBP, catalog number: 7003 ) stored at room temperature
  37. SYBRTM Safe DNA Gel Stain (Thermo Fisher Scientific, Invitrogen, catalog number: S33102 ) stored at 4 °C
  38. Complete parasite culture medium (see Recipes)
  39. Ethanol 75% (see Recipes)
  40. 10x TBE (see Recipes)
  41. 2 mM EDTA (see Recipes)
  42. 1x TBE (see Recipes)
  43. 1x TBE-SDS 2% (see Recipes)
  44. 1 kb DNA Ladder (50 µg/ml) (see Recipes)

Equipment

  1. Autoclaved glass bottles (SCHOTT DURAN)
  2. -80 °C freezer
  3. FormaTM Steri-CycleTM CO2 Incubator at 26 °C (Thermo Fisher Scientific, catalog number: 371 )
  4. Pipette controller (INTEGRA, catalog number: 155017 )
  5. Pipettes 1,000 µl, 200 µl, 20 µl (Gilson®)
  6. Pipette 10 µl (Discovery Comfort)
  7. Laminar flow hood parasite culture (SafeFAST Premium)
  8. Chemical hood
  9. Centrifuge 5810 R (Eppendorf) at room temperature and set here at 24 °C
  10. Centrifuge MICRO STAR 17 (VWR) kept in a cold room at 4 °C
  11. NanoDropTM ND-1000 Spectrophotometer (ThermoFisher Scientific)
  12. Water bath (JULABO GmbH)
  13. Balance (NewClassic MF, MS4002SDR, METTLER TOLEDO)
  14. Ultra ClearTM (BLANC-LABO) system
    Note: The Ultra ClearTM (BLANC-LABO) system delivers Ultra-pure DNase/RNase-free water.
  15. Mini-gel migration system “home-made” (see Figure 2)
  16. Electrophoresis Power Supply (GIBCO BRL, Life Technologies, ST305) and power cables
  17. Microwave (FAR)
  18. Blue LED transilluminator (Dark Reader DR-88X, Clare Chemical Research)
  19. Camera (Computar iAi CVAA5 IR No C510390B RoHS)
  20. Video Graphic Printer (SONY, UP-895CE)

Procedure

The steps of the RNA extraction (A) and purification (B) are summarized in Figure 1.



Figure 1. Steps of RNA extraction and purification. This diagram represents the steps of RNA extraction (A) and purification (B). Certain illustrations are from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Unported License. https://smart.servier.com/.


  1. Total RNA extraction (not DNA-free) from Leishmania parasites (see Figure 1)
    Note: The strains are not available commercially. They are derived from Lgy M4147 (MHOM/BR/75/M4147).
    1. Under a laminar flow hood, collect 5 ml of a 10 ml stationary phase parasite culture (day 6 of culture, for a detailed protocol of culture, see pages 3 and 4 of the Bio-protocol by Reverte and Fasel, 2019) in a 50 ml tube.
      Note: The authors recommend to use a 50 ml tube even for 5 ml parasites, as discarding the medium from the pellet (at Step A3) is easier with a 50 ml tube.
    2. Centrifuge at 2,000 x g for 10 min at 24 °C.
    3. Under a laminar flow hood, discard the medium and resuspend the pellet with 1 ml of DPBS 1x.
    4. Under a laminar flow hood, transfer to a 1.5 ml tube.
    5. Centrifuge at 2,500 x g for 10 min at 4 °C.
    6. Under a laminar flow hood, discard the supernatant.
    7. Resuspend and homogenize the pellet with 1 volume of TRI® reagent (1 ml) under chemical hood, in order to extract RNA from the parasites. At this step, the sample can be kept for a few weeks at -80 °C.

  2. Total RNA purification (not DNA-free) from Leishmania parasites (see Figure 1)
    Note: The following protocol is adapted from the protocol of TRI® reagent provided by the manufacturer.
    1. Clean the bench and the pipettes with RNase AWAYTM reagent.
    2. If kept at -80 °C, thaw the sample and let equilibrate to room temperature (RT) for 5 min.
    3. Under chemical hood, add 1/5 volume of chloroform (200 µl).
    4. Mix vigorously and wait for 2 min at room temperature.
    5. Centrifuge at 12,000 x g for 15 min at 4 °C.
    6. Transfer the upper aqueous phase (approximately 500 µl) into a new 1.5 ml tube.
    7. Add the same volume of isopropanol (here 500 µl) and mix, wait for 5 min at room temperature.
      Note: At this step, the purification procedure can be stopped and the tube can be stored at -20 °C overnight.
    8. Centrifuge at 12,000 x g, for 20 min, at 4 °C.
    9. Discard the supernatant and add 1 volume of Ethanol 75% (1 ml) to the pellet.
    10. Centrifuge at 7,500 x g, for 10 min, at 4 °C and discard the supernatant.
    11. Let the pellet dry at room temperature under the chemical hood for approximately 10 min.
      Note: Do not over dry the pellet as it may be difficult to dissolve.
    12. Add 25 µl UltraPureTM DNase/RNase-free Distilled Water and vortex briefly to dissolve the pellet at room temperature.
    13. Put the tube on ice and measure RNA concentration with 1.5 µl RNA with NanoDropTM. Check that the A260/A280 and A260/A230 ratios are of good quality (not contaminated).
      Note: Make the blank of the NanoDropTM with 1.5 µl of UltraPureTM DNase/RNase-free Distilled Water.
    14. RNA can be kept at -80 °C for long storage.
      Note: Repeated freeze and thaw can decrease the concentration of the RNA and fresh RNA can give better results.

  3. Enzymatic digestion of total RNA (not DNA-free) from Leishmania parasites: degradation of DNA by DNase I, and single-stranded DNA or RNA by Nuclease S1
    1. Clean the bench and the pipettes with RNase AWAYTM reagent.
    2. Warm up the water bath to 37 °C.
    3. Thaw parasite-isolated total RNA (if frozen) and keep on ice.
      Note: If the RNA is frozen, measure RNA concentration with NanoDropTM after it thaws.
    4. In a new 1.5 ml tube, add between 5 and 50 µg of RNA, on ice.
    5. For 1 reaction, digest the total RNA (not DNA-free) with 60 U of Nuclease S1 in Buffer S1 1x and with 10 U of DNase I in UltraPureTM DNase/RNase-free Distilled Water.
      Note: In a new 1.5 ml tube on ice, prepare the enzymatic mix for several reactions: mix DNase I, Nuclease S1, Buffer S1 diluted to 1x and UltraPureTM DNase/RNase-free Distilled Water. Then dispense on RNA. This enzymatic mix has been tested on the range: 5 to 50 µg of RNA.
    6. Prepare an undigested negative control for each sample: in a new 1.5 ml tube, on ice, add between 2 and 5 µg of RNA and dilute in UltraPureTM DNase/RNase-free Distilled Water (final volume of 10 µl).
      Note: Different quantities of RNA between digested and undigested samples have been used to avoid huge differences of exposure times between them when acquiring pictures of the gel electrophoresis.
    7. Vortex briefly and spin down.
    8. Incubate the tubes containing RNA and enzymatic mix, and negative control (without enzymatic mix), in the water bath at 37 °C for 1 h.
      Note: During this incubation time, the agarose gel can be prepared.
    9. At the end of the incubation, transfer the tubes with the digested RNA and the undigested negative control on ice.
      Note: At this step, the samples can be kept at -80 °C for several weeks.

  4. Visualization of viral dsRNA by migration on mini-gel electrophoresis
    1. Rinse the autoclaved glass bottles needed with Ultra-pure DNase/RNase-free water, type Ultra ClearTM.
    2. Prepare 1x TBE solution (Recipe 5) in an autoclaved glass bottle.
    3. Prepare 1x TBE containing 2% SDS solution (Recipe 6) in an autoclaved glass bottle.
    4. Clean the material for mini-gel electrophoresis (tank, spacers and comb) with 1x TBE containing 2% SDS solution, then rinse very well with Ultra-pure DNase/RNase-free water, type Ultra ClearTM and with RNase AWAYTM reagent.
    5. Prepare 0.8% agarose gel.
      1. Weigh agarose powder in an autoclaved glass bottle: 0.4 g for 1 mini-gel.
      2. Add 1x TBE: 50 ml for 1 mini-gel.
      3. Heat with a microwave until agarose is homogeneously dissolved.
        Note: Monitor the aspect of the mixture and be careful of the high temperature.
      4. Cool down at room temperature for a few min.
    6. Mount the mini-gel electrophoresis (see Figure 2).


      Figure 2. Mini-gel migration system “home-made” for gel electrophoresis. This figure represents the different pieces to assemble before putting the agarose gel.

    7. Add SYBRTM Safe to agarose gel (0.5 µl for 1 mini-gel), shake well but without making bubbles and fill the electrophoresis tank.
    8. Remove potential bubbles with a tip and let the agarose gel polymerize for 30-45 min at room temperature.
    9. Pour 1x TBE in the electrophoresis tank (75 ml for 1 mini-gel) and carefully remove the spacers and the comb.
    10. Add 6x Gel Loading Dye to the samples: the digested RNA and the undigested negative control in order to have Gel Loading Dye at 1x.
    11. Load the wells with 10-25 µl of each sample and 2-4 µl of 1 kb DNA Ladder (50 µg/ml).
    12. Connect the electrophoresis tank to the power supply with cables: the top electrode is connected to the negative pole and the bottom electrode to the positive pole.
    13. Switch on the power supply: run the gel at 80-100 volts for 45-60 min, until the Gel Loading Dye has reached the ¾ of the mini-gel.
    14. Switch off the power supply and disconnect the electrophoresis tank.
    15. Transfer the gel to a transilluminator coupled to camera and printer, adjust the zoom, the focus and the exposure time, then take a picture of the mini-gel (see Figure 3).
    16. Observe and analyse the picture of the gel electrophoresis (see Data analysis).

Data analysis

In the figure of the gel electrophoresis, in the digested RNA (+) lanes (see Figure 3), an upper band at around 5 kb (see blue arrow) is observed in the digested RNA sample from LRV+ parasites, and not in the digested RNA sample from LRV- parasites. The theoretical size of LRV is 5.3 kb. The DNase I degrades dsDNA and ssDNA of the RNA samples that were not free from DNA, whilst the Nuclease S1 degrades single-stranded nucleic acids, such as ssRNA and ssDNA. In Figure 3, the viral dsRNA from LRV (see blue arrow) can be visualized after enzymatic digestions because DNase I and Nuclease S1 do not degrade dsRNA. In the undigested negative controls (-), 3 major bands between 1.5 and 2 kb are observed, they represent the 3 ribosomal RNAs (rRNA), which are ssRNA. The dsRNA at around 5 kb is also observed (see blue arrow). There can also be other bands corresponding to contaminant DNA.
Note: To go further, it is also possible to treat with RNase III enzyme for 20 min at 37 °C. The RNase III degrades dsRNA. Following gel electrophoresis, the band at around 5 kb will no longer be visible.


Figure 3. Image of the gel at the end of electrophoresis. The Ladder represents the 1 kb DNA Ladder loaded into the first well on the left. The scale in kilobases (kb) is indicated (from 10 to 0.5 kb). The negative controls without enzymatic digestions are represented by (-). The samples, which are digested, are indicated with (+). The black dividing line (between LgyLRV1- samples and LgyLRV1+ samples) represents where the splice junction has been made. The blue arrow indicates the band of the dsRNA. Lgy: Leishmania guyanensis; LRV1: Leishmania RNA Virus; LRV1-: negative for LRV presence; LRV1+: positive for LRV presence.

Recipes

  1. Complete parasite culture medium (500 ml)
    1. Remove 111 ml from the 500 ml Schneider’s Drosophila Medium.
    2. Then add 100 ml heat-inactivated FBS (see Recipes from Bio-protocol by Reverte and Fasel, 2019), 5 ml HEPES, 5 ml P/S, 250 μl Biopterin (see Recipes from Bio-protocol by Reverte and Fasel, 2019), 1 ml Hemin-folate (see Recipes from Bio-protocol by Reverte and Fasel, 2019).
    3. Filter the complete parasite culture medium using Vacuum Filtration 500–Filtermax, aliquot to 50 ml tubes and store at 4 °C.
  2. Ethanol 75%
    1. For 50 ml, in a 50 ml tube, add 17.5 ml Ultra-pure DNase/RNase-free water, type Ultra ClearTM with a 25 ml pipette connected to a pipette controller.
    2. Then add 37.5 ml Ethanol absolute.
    3. Store at 4 °C.
  3. 10x TBE (1 L)
    1. Add 108 g Tris(hydroxymethyl)aminomethane, 55 g boric acid and 2 mM EDTA (Recipe 4)
    2. Add water to 1 L
    3. Store at room temperature
  4. 2 mM EDTA (1 L)
    1. Add 0.74446 g EDTA powder
    2. Add water to 1 L
    3. Store at room temperature
  5. 1x TBE (1 L)
    1. Add 100 ml 10x TBE (Recipe 3) and 900 ml water
    2. Store at room temperature
  6. 1x TBE-SDS 2%
    1. For 250 ml, add 25 ml SDS 20% and 225 ml 1x TBE (Recipe 5)
    2. Store at room temperature
  7. 1 kb DNA Ladder (50 µg/ml)
    1. For 240 µl, in a 1.5 ml tube, on ice, add 176 µl of UltraPureTM DNase/RNase-free Distilled Water
    2. Add 40 µl of 6x Gel Loading Dye and add 24 μl of 1 kb DNA Ladder at 500 µg/ml
    3. Store at 4 °C

Acknowledgments

We thank Slavica Masina and Tiia Snäkä for critical reading of the manuscript; Marta Reverte for helping with several catalog numbers already referenced (see Bio-protocol by Reverte and Fasel, 2019); Chantal Desponds and Florence Prével for providing technical advice on protocol development. This research was supported by FNS grant No 310030_173180 and No IZRJZ3_164176/1 to NF. This protocol was adapted from the following original research papers by Ives et al., 2011; Zangger et al., 2013 and 2014; Grybchuk et al., 2018.

Competing interests

The authors declare no competing interests.

References

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  18. Zangger, H., Ronet, C., Desponds, C., Kuhlmann, F. M., Robinson, J., Hartley, M. A., Prevel, F., Castiglioni, P., Pratlong, F., Bastien, P., Müller, N., Parmentier, L., Saravia, N. G., Beverley, S. M. and Fasel, N. (2013). Detection of Leishmania RNA virus in Leishmania parasites. PLoS Negl Trop Dis 7(1): e2006.

简介

[摘要 ] 原生动物寄生虫中发现了许多RNA病毒,它们可能导致更严重的病理或治疗失败。对于病毒双链RNA(dsRNA)的检测,有序列依赖性和非依赖性方法,例如定量实时PCR和免疫荧光,斑点印迹,ELISA或测序。此处介绍的技术是与序列无关的,并且在以下协议中进行了详细说明,以利什曼原虫(Legymania guyanensis)(Lgy )中的利什曼原虫RNA病毒(LRV)为例 概括地说,该协议分为四个主要步骤:从寄生虫中提取RNA,RNA纯化,使用DNase I和Nuclease S1进行图解消化以及通过凝胶电泳进行可视化。该方法可用于检测其他病毒dsRNA它提供了一个额外的工具,可以对先前引用的其他技术进行补充,并且很容易实现。

[背景 ] 广泛的多样性RNA病毒中存在的原生动物寄生虫一直都有详细记载(王和王,1991;戈什等人,2011;桑戈等人。2014;碱液等人,2016年Akopyants 等人2016 ; Fernandez- Presas 等人,2017; Grybchuk 。等人。,2018)。此外,这些病毒已经被描述为潜在的毒力因子(Fichorova 等人,2013; EL- Gayar 。等人,2016; 拉特等。,2019)特别值得注意的,存在的内共生体。利什曼原虫RNA病毒(LRV),A 整体病毒科的双链RNA(dsRNA的)病毒,在利什曼原虫Guyanensis (LGY )加剧利什曼病病(艾夫斯等人,2011;罗西的Et的Al ,2017),费弗斯转移通过诱导白介素-17(哈特利等人,2016),并且也增加了治疗失败的风险(Adaui 等人,2016; Bourreau 。等人,2016; 维埃拉-贡萨尔维斯等。,2019)。这表明在寄生虫中检测病毒dsRNA 的重要性。与quan相比,此处介绍的技术是与序列无关的 实时荧光定量PCR技术,因此可以广泛应用于RNA病毒。该dsRNA检测方案也可以(建议)用于确认(或与之确认)其他独立于序列或依赖于序列的检测方法例如点印迹或PCR(Zangger 等,2013)。该技术与LRV结合使用,但它可用于其他RNA病毒和其他寄生虫(Grybchuk 等,2018)。

关键字:dsRNA, 病毒, 寄生虫, 利什曼原虫RNA病毒, 检测, DNase I, Nuclease S1

材料和试剂


 


1. Eco Nitrile PF 250手套(ecoSHIELD TM )      


2. 过滤嘴低保留量1,000 µl,200 µl,20 µl(ClearLine ,目录号分别为713118、713117和713115)      


3.10 µl加长过滤嘴(海王星科学,目录号:BT10XL)      


4. 组织培养瓶25版“放空”(TPP ® ,目录号:90025)      


5. 真空过滤500 “快速” - Filtermax ,PES膜0.22 Myuemu 孔径(TPP ® ,目录号:99500)      


6. 微量1.5毫升管(Corning公司,爱思进®,目录数目:MCT-175-C)      


7. 聚丙烯锥形50 ml离心管(TPP Techno Plastic Products,货号:91050)      


8. 2毫升血清移液管(FALCON ® ,目录号:357507)      


9. 5毫升血清移液管(SARSTEDT,目录号:86.1253.001)      


10. 10毫升血清移液管(SARSTEDT,目录号:86.1254.001)   


11. 25毫升血清移液器(SARSTEDT,目录号:86.1685.001)   


12.将Dulbecco的磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM ,目录号:14040091)储存在4°C下   


13. 施耐德果蝇培养基w:L-谷氨酰胺和0.40 g / L NaHCO 3 (PAN TM BIOTECH,目录号:P04-91500)储存于4°C   


14. 胎儿牛血清(FBS)(热Fisher Scientific公司,Gibco公司TM ,目录号:10270106),已发送在-20℃下,然后热灭活(见配方从生物协议通过Reverte 而且法泽尔,2019 ),分注于50毫升试管,并储存在-20°C   


15. HEPES Buffer 1 M(BioConcept ,目录号:5-31F00-H)在4°C储存   


16. 青霉素-链霉素(P / S)溶液(10,000 IU / ml的P和10毫克/毫升S)(Bioconcept ,目录数目:4-01F00-H),发送的在-20℃下,然后将储存在4℃下用于短期存储   


17. Hemin BioXtra ,购自Porcine(Sigma-Aldrich,目录号:51280),在4°C下储存   


18. 叶酸(Fluka TM ,目录号:47620)在4°C储存   


19. 6-生物蝶呤(Sigma-Aldrich公司,目录号的:B2517)存储在-20℃下   


20. TRI ® 试剂(分子研究中心,MRC,目录号码:TR118,500毫升)中的保存在4 ℃下   


21. 室温下保存的氯仿(Thermo Fisher Scientific,Fisher Chemical,目录号:C / 4960/15)   


22. 异丙醇(Thermo Fisher Scientific,Fisher Chemical,目录号:P / 7490/17)在室温下保存   


23. 在室温下储存的无水乙醇(Thermo Fisher Scientific,Fisher Chemical,目录号:E / 0650DF / 17)   


24. UltraPure TM 不含DNase / RNase的蒸馏水(Thermo Fisher Scientific,Invitrogen TM ,目录号:10977-035 ),在室温下运输,在打开前在4°C下储存,等分至1.5 ml管中并在-20℃下储存°C   


25. DNase I,不含RNase的10 U / µl ,在-20°C下储存的小牛胸腺DNA (Roche,目录号:10 776 785 001)的活性为0.2 U / µg。   


26. 核酸酶S1(#E576)(Promega ,目录号:M5761)储存在-20°C   


注意:请注意以下事实:浓度对每个试管都是特定的,并且由制造商在试管上标明。


27. 缓冲液S1(#E578)(Promega ,目录号:M577A)储存在-20°C   


注:牛逼他老参考在7.4倍,但现在S1核酸酶反应缓冲液中被设置在10X 。


28. 1 kb的DNA梯子500 微克/毫升(NEB,目录号的:N3232S)存储在4℃下   


29. 6X凝胶上样染料(紫色),无SDS (NEB,目录号:B7025)储存在4℃;提供与所述1 kb的DNA梯   


30. 标准琼脂糖    –常规凝胶电泳的LE型(BioConcept Ltd,经修正,目录号:7-01P02-R)在室温下保存


31. 三(羟甲基)氨基甲烷,室温保存的分子生物学(Biosolve ,目录号:20092391)   


32. 室温保存的硼酸(结晶粉末/电泳级)(Fisher BioReagents ,目录号:BP168-1)   


33. EDTA ,Ithylenediaminetetraacetic酸,二钠盐二水合物(结晶性粉末/电泳级)(费舍尔BioReagents公司,目录编号:BP120-1)储存在室温Temperat .. [温度茜   


34. SDS ,钠十二烷基,小号Olution 20 Pasento纯(APPLICHEM PANREAC ,ITW试剂,目录编号:A3942,1000)在室温下存放   


35. 超纯水,类型Ultra Clear TM (BLANC-LABO),在室温下储存   


36. 核糖核酸酶AWAY TM (分子生物产品TM ,MBP,目录编号:7003)在室温下存放   


37. SYBR TM 安全DNA凝胶染色(热Fisher Scientific公司,Invitrogen公司,目录号的:S33102)的保存在4℃下   


38. 完整的寄生虫培养培养基(参见配方小号)   


39. 乙醇75%(请参阅食谱s )   


40. 10X TBE(见配方小号)   


41. 2mM EDTA的(见配方小号)   


42. 1X TBE(见配方小号)   


43.1 TBE - SDS 2%(请参阅配方s )   


44.1 kb DNA阶梯(50 µg / ml)(请参阅第s章)   


 


配套设备


 


高压灭菌玻璃瓶(SCHOTT DURAN)
-80°C冷冻室
FORMA TM 免缝周期TM CO 2 培养箱在26℃(热˚F Isher科学,目录号码:371)
移液器控制器(INTEGRA,目录号:155017)
1000移液器MYU L,200 MYU L,20 MYU L(吉尔森® )
移液管10 MYU L(发现舒适)
层流罩寄生虫培养(SafeFAST Premium)
化学罩
在室温下将5810 R(Eppendorf)离心机设置在24°C
MICRO STAR 17(VWR)离心机保持在4°C的冷藏室中
NanoDrop TM ND-1000分光光度计(ThermoFisher Scientific)
水浴(JULABO GmbH )
天平(NewClassic MF,MS4002SDR,梅特勒-托利多)
Ultra Clear TM (BLANC-LABO)系统
注意:Ultra Clear TM (BLANC-LABO)系统提供超纯DNase / RNase的水。


微型凝胶迁移系统“自制”(见图2 )
电泳电源(GIBCO BRL,Life Technologies,ST305)和电源线
微波(FAR )
蓝色LED透照器(Dark Reader DR-88X,Clare Chemical Research )
相机(Computar iAi CVAA5 IR编号C510390B RoHS)
视频图形打印机(SONY,UP-895CE)
 


程序


 


图1总结了RNA提取(A)和纯化(B)的步骤。


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-5-05 \ 3598--1902875--1337 Nicolas Fasel 825328 \ Figs jpg \图1-updated.jpg


1.图步骤RNA提取和纯化。该图表示了步骤RNA提取(A)和纯化(B)某些插图来自。施维雅医学技术,许可经Creative共同署名3.0 Unported 许可。HTTPS://智能.servier.com /。


从利什曼原虫中提取总RNA(并非不含DNA)(见图1)
注意:这些菌株无法从市场上买到,它们来自Lgy M4147(MHOM / BR / 75 / M4147)。


在层流罩下,在50毫升中收集5毫升10毫升固定相寄生虫培养物(培养的第6天,有关详细的培养方案,请参见《生物协议》第3和4页,Reverte 和Fasel ,2019年)毫升管。
注:作者建议使用50毫升管连服5种毫升的寄生虫,如丢弃介质从颗粒(在小号Tep的A3 )较易于50mL试管中。


在24°C下以2,000 x g 离心10分钟。
在层流罩下,弃去培养基,并用1 ml DPBS 1x重悬沉淀。
在层流罩下,转移至1.5 ml管中。
在4°C下以2500 xg离心10 m 。
在层流罩下,丢弃骨料。
和均质化再中止圆球1卷TRI ® 试剂(1毫升)中的化学罩,以提取RNA从寄生虫。在这一步中,样品可以保持几个星期在-80℃
 


从利什曼原虫寄生虫中纯化总RNA(并非不含DNA)(见图1)
注意:以下协议是改编自协议TRI ® 提供由试剂制造商。


用RNase AWAY TM 试剂清洁工作台和移液器。
如果保持在-80°C,则将样品解冻,并使其平衡至室温(RT)5分钟。
在化学保护罩下,加入1/5体积的氯仿(200 µl )。
剧烈混合,然后在室温下等待2分钟。
在4°C下以12,000 xg离心15分钟。
将上层水相(约500 µl )转移到新的1.5 ml管中。
加入相同体积的异丙醇(此处为500 µl)并混合,在室温下等待5分钟。注意:在此步骤中,可以停止纯化过程,并将试管在-20°C下保存过夜。
在4°C下以12,000 xg离心20分钟。
              丢弃补品,并向沉淀物中加入1体积的75%乙醇(1毫升)。
              在7离心机,500 ×g下,10分钟,在4℃并弃去上清液。
让沉淀在室温下在化学通风橱中干燥约10分钟。
注意:不要过度干燥沉淀物,因为它可能很难溶解。


加入25 µl UltraPure TM DNase /不含RNase的蒸馏水,并短暂涡旋以在室温下溶解沉淀。
将试管放在冰上,并用NanoDrop TM 测量1.5 µl RNA的RNA浓度。检查A 260 / A 280 和A 260 / A 230的比率质量是否良好(未污染)。
注意:请的空白的纳米滴TM 采用1.5 Myueru 超纯TM DNA酶/ RNA酶的蒸馏水。


RNA可以保存在-80°C以便长期保存。
注意:反复冷冻和融化可以降低RNA的浓度,而新鲜RNA可以提供更好的结果。


 


利什曼原虫寄生虫对总RNA(不是不含DNA)的酶消化:DNase I降解DNA,Nucleus e S1 降解单链DNA或RNA
用RNase AWAY TM 试剂清洁工作台和移液器。
将水浴加热至37°C。
解冻寄生虫分离的总RNA(如果冷冻),并置于冰上。
注意:如果RNA是冷冻的,则在解冻后用NanoDrop TM 测量RNA浓度。


在新的1.5 ml试管中,在冰上添加5至50 µg RNA。
对于1个反应,请在缓冲液S1 1x中用60 U核酸酶S1和在UltraPure TM 不含DNase / RNase的蒸馏水中用10 U DNase I消化总RNA(非DNA)。
注意:在新的1.5 ml冰试管中,为几种反应准备理论混合物:将DNase I,核酸酶S1,稀释至1x的Buffer S1和UltraPure TM DNase /不含RNase的蒸馏水混合,然后分配RNA。已在以下范围内进行了测试:5至50 µg RNA。


为每个样品准备一个未消化的阴性对照:在一个新的1.5 ml试管中,在冰上,添加2至5 µg RNA,并在UltraPure TM DNase /不含RNase的蒸馏水中稀释(终体积为10 µl)。
注意:在获取凝胶电泳图片时,已消化和未消化样品之间使用了不同数量的RNA,以避免它们之间的暴露时间存在巨大差异。


短暂涡旋并旋转。
在37°C水浴中将装有RNA和有目的混合物以及阴性对照(无主要混合物)的试管孵育1小时。
注意:在此孵育时间内,可以制备琼脂糖凝胶。


孵育结束时,将含有消化的RNA和未消化的阴性对照的试管转移到冰上。
注意:在此步骤中,样品可以在-80°C下保持数周。


 


的dsRNA病毒可视化通过迁移在迷你凝胶电泳
用超纯DNase /无RNase的水冲洗所需的高压灭菌玻璃瓶,类型为Ultra Clear TM 。
在高压灭菌的玻璃瓶中准备1x TBE溶液(配方5)。
在高压灭菌的玻璃瓶中准备1x含2%SDS 溶液的TBE (配方6 )。
用1x含2%SDS溶液的TBE 清洁用于微型凝胶电泳的材料(槽,垫片和梳子),然后用超纯DNase /无RNase的水,Ultra Clear TM 类型和RNase AWAY TM 试剂彻底清洗。
准备0.8%琼脂糖凝胶。
在高压灭菌的玻璃瓶中称量琼脂糖粉末:0.4克(1毫升迷你凝胶)。
加入1x TBE:50毫升(1毫升迷你凝胶)。
微波加热均匀加热。
注意:监控混合物的外观并注意高温。


在室温下冷却几分钟。
安装小凝胶电泳(参见图2)。
 


D:\重新格式化\ 2020-2-7 \ 1902875--1337 Nicolas Fasel 825328 \图jpg \图2.jpg


图2.凝胶电泳的“自制”微型凝胶迁移系统此图表示在放置琼脂糖凝胶之前要组装的不同片段。


 


SYBR添加TM 安全琼脂糖凝胶(0.5 MYU L关于1小型凝胶),摇匀但不使气泡和填充电泳槽。
用尖端除去潜在的气泡,让琼脂糖凝胶在室温下聚合30-45分钟。
将1x TBE倒入电泳槽中(75 ml用于1个微型凝胶),并小心地移开垫片和梳子。
向样品中添加6x凝胶上样染料:消化的RNA和未消化的阴性对照,以使凝胶上样染料达到1x。
的井用负载10-25 MYU L各自的样品和2-4 MYU 模式L 1个kb的DNA梯(50 MYU 微克/毫升)。
用电缆将电泳槽连接到电源:顶部电极连接到负极,底部电极连接到正极。
接通电源:在80-100伏特下运行凝胶45-60分钟,直到凝胶负载染料达到微型凝胶的¾。
关闭电源并断开电泳槽。
将凝胶转移到与照相机和打印机相连的透照器上,调节变焦,聚焦和曝光时间,然后拍摄微型凝胶的照片(见图3)。
观察并分析凝胶电泳图(请参阅数据分析)。
 


资料分析


 


在凝胶电泳图中,在消化的RNA(+)泳道中(参见图3),在从LRV + 寄生虫消化的RNA样品中观察到约5 kb的上带(见蓝色箭头),而不是在消化RNA样品从轻轨- ..寄生虫理论大小LRV为5.3 kb的DNA酶我性能降级双链DNA单链DNA和RNA样品的未摆脱的DNA,而S1核酸酶降解单链核酸,如单链RNA 和在图3中,由于DNase I和核酸酶S1不会降解dsRNA,因此在合成后可以看到LRV的病毒dsRNA(见蓝色箭头)在未消化的阴性对照(-)中,有1.5至2 kb的3条主要条带观察到,它们代表了3个核糖体RNA(rRNA ),即ssRNA。还观察到了约5 kb的dsRNA(见蓝色箭头)。还可能存在与污染物DNA相对应的其他条带。


注意:进一步,也可以在37°C下用RNase III酶处理2 0分钟,RNase III降解dsRNA,凝胶电泳后,约5 kb处的条带将不再可见。


 


D:\重新格式化\ 2020-2-7 \ 1902875--1337 Nicolas Fasel 825328 \图jpg \图3.jpg


图3.电泳结束时的凝胶图像。梯形图代表加载到左侧第一个孔中的1 kb DNA梯形图。标度以千碱基为单位(kb)(从10到0.5 kb)。阴性对照如果没有酶消化来表示通过( - )。该样本为基础,消化,以表示(Tasu)黑分野(间LGY LRV1-样品以及LGY 。LRV1 Tasu样品)代表的剪接点已经取得的蓝箭头指示dsRNA 的条带。Lgy :利什曼原虫(Leishmania guyanensis); LRV1:利什曼原虫RNA病毒; LRV1-:LRV存在阴性; LRV1 +:LRV存在阳性。


 


菜谱


 


完全寄生虫培养基(500 ml )
从500 毫升施耐德果蝇培养基中取出111毫升。
在接着该加载100毫升热灭活的FBS(见配方从生物协议通过Reverte 和法泽尔,2019 ),将5ml HEPES,加入5ml P / S,250 微升生物蝶呤(见配方从生物协议通过Reverte 和法泽尔,2019)加入1ml氯化血红素叶酸(见配方从生物协议通过Reverte 和法泽尔,2019)。
使用真空过滤500– Filtermax 过滤完整的寄生虫培养基,等分至50 ml管中并在4°C下储存。
乙醇75%
对于50毫升,在50毫升试管中,加入17.5毫升超纯DNase /无RNase的水,键入Ultra Clear TM ,将25毫升移液器连接到移液器控制器上。
然后加入37.5毫升无水乙醇。
储存在4℃。
10x TBE (1公升)
甲DD108克三(羟甲基)氨基甲烷到55克硼酸和2mM EDTA(配方4 )
加水至1 升
室温保存
2 mM EDTA (1升)
甲DD 0.74446ģEDTA粉末
加水至1 升
室温保存
1个TBE (1公升)
甲DD100毫升10X TBE(配方3)和900毫升的水
室温保存
1x TBE - SDS 2%
对于250毫升,添加25毫升20%SDS和225毫升1x TBE(配方5)
室温保存
1 kb DNA阶梯(50 µg / ml)
对于24 0 µl,在1.5 ml管中,在冰上,添加176 µl UltraPure TM DNase /无RNase蒸馏水
40 Myueru添加6X凝胶上样染料和添加24 Myueru ö f 1的kb的DNA梯子500 Myug / ml的
储存在4°C
 


 


致谢


 


我们感谢斯拉维卡西娜和Tiia Snaka 对于批判性阅读的手稿;玛塔Reverte 帮助与已经引用(看到几个目录号生物协议通过Reverte 而法塞尔2019年);尚塔尔Desponds而佛罗伦萨Prevel 提供技术咨询,协议开发。这项研究是支持通过FNS批准号310030_173180并没有IZRJZ3_164176 / 1 NF 此协议是改编自后面原始研究论文通过艾夫斯等,2011;桑戈等人,2013 和2014; Grybchuk 。等人,2018 。


 


竞争利益


 


作者宣称没有利益冲突。


 


参考文献


 


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引用:Isorce, N. and Fasel, N. (2020). Viral Double-Stranded RNA Detection by DNase I and Nuclease S1 digestions in Leishmania parasites. Bio-protocol 10(9): e3598. DOI: 10.21769/BioProtoc.3598.
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