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Formation of viral particles and packaging of genomic retroviral RNA into these particles are important steps in the late phase of the viral replication cycle. The efficiency of the incorporation of viral or cellular RNAs into viral particles can be studied using a quantitative Reverse Transcriptase-PCR (RT-qPCR)-based approach. After isolation of cytoplasmic RNA from either infected or transfected cells and extraction of virus particle-associated RNA, specific RNA levels present in both fractions are determined. The ratio of virion-associated and cytoplasmic RNA defines the encapsidation efficiency (Brandt et al., 2007; Blissenbach et al., 2010; Grewe et al., 2012).

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Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR
通过量化分析RT-qPCR将逆转录病毒RNA整合到病毒颗粒中

微生物学 > 微生物遗传学 > RNA > qRT-PCR
作者: Bianca Hoffmann
Bianca HoffmannAffiliation: Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
Bio-protocol author page: a446
 and Bastian Grewe
Bastian GreweAffiliation: Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
For correspondence: bastian.grewe@rub.de
Bio-protocol author page: a371
Vol 3, Iss 8, 4/20/2013, 6555 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.684

[Abstract] Formation of viral particles and packaging of genomic retroviral RNA into these particles are important steps in the late phase of the viral replication cycle. The efficiency of the incorporation of viral or cellular RNAs into viral particles can be studied using a quantitative Reverse Transcriptase-PCR (RT-qPCR)-based approach. After isolation of cytoplasmic RNA from either infected or transfected cells and extraction of virus particle-associated RNA, specific RNA levels present in both fractions are determined. The ratio of virion-associated and cytoplasmic RNA defines the encapsidation efficiency (Brandt et al., 2007; Blissenbach et al., 2010; Grewe et al., 2012).
Keywords: HIV(艾滋病咨询门诊), Encapsidation(包装), Packaging(包装), RT-PCR(RT-PCR), Viral Particles(病毒颗粒)

[Abstract] 病毒颗粒的形成和基因组逆转录病毒RNA包装到这些颗粒中是病毒复制周期晚期的重要步骤。 可以使用定量逆转录酶-PCR(RT-qPCR)为基础的方法研究将病毒或细胞RNA掺入病毒颗粒中的效率。 在从感染的或转染的细胞分离细胞质RNA并提取病毒颗粒相关的RNA后,测定两种级分中存在的特异性RNA水平。 病毒体相关的和细胞质RNA的比率定义了衣壳化效率(Brandt等人,2007; Blissenbach等人,2010; Grewe等人,/em>,2012)。

Materials and Reagents

I. Cell harvesting and ultracentrifugation

  1. Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Gibco®, catalog number: 41965-039 )
  2. Fetal calf serum (FBS) (Life Technologies, Gibco®, catalog number: 10108-165 )
  3. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 14200-067 )
  4. Trypsin (Biochrom)
  5. Penicillin/Streptomycin (Life Technologies, Gibco®)
  6. 0.45 μm filter (Sarstedt)
  7. Sucrose (Biomol)
  8. Ultracentrifugation tubes (17 ml) (Herolab)

II. Cell fractionation and RNA isolation

  1. Dithiothreitol (DTT) solution (1 M) (AppliChem GmbH, catalog number: A3668,0050 )
  2. RiboLock RNase Inhibitor (40 U/μl) (Thermo Fisher Scientific, catalog number: E00384 )
  3. Ethanol >99.8% (Sigma-Aldrich)
  4. QIAshredder Spin Columns (QIAGEN, catalog number: 79654 )
  5. RNeasy Kit (QIAGEN, catalog number: 74106 )
  6. QIAmp Viral RNA Kit (QIAGEN, catalog number: 52906 )
  7. TURBO DNA-free Kit (Life Technologies, Ambion®, catalog number: AM1907 )
  8. Nuclease-free water (B Braun)
  9. Quant-iT RNA Assay Kit (Life Technologies, InvitrogenTM, catalog number: Q-33140 )

III. RT-qPCR

  1. QuantiTect Probe RT-PCR Kit (includes Reverse Transcriptase) (QIAGEN, catalog number: 204443 )
  2. SYBR Green (Life Technologies, InvitrogenTM)
  3. Primers sense and antisense (Biomers)
  4. Isolated RNA
  5. TOPO TA Cloning Kit (Life Technologies, InvitrogenTM, catalog number: 45-0640 )
  6. AmpliScribe-T7/Sp6 High Yield Transcription Kit (Epicentre, catalog number: AS2607 )
  7. RNA standards

IV. Other materials

  1. HIV-1 DIY p24 sandwich ELISA Kit 2 (Aalto Bio Reagents)

Equipment

  1. Heraeus Multifuge X3R Centrifuge (Thermo Fisher Scientific)
  2. Centrifuge 5417R (Eppendorf)
  3. Sorvall WX Ultra 100 Ultracentrifuge (Thermo Fisher Scientific)
  4. Qubit Fluorometer (Life Technologies, InvitrogenTM, catalog number: Q32857 )
  5. Rotor-Gene Q (QIAGEN, catalog number: 9001620 )
  6. Strip tubes 0.1 ml (QIAGEN)
  7. 15 ml tubes (Sarstedt)
  8. 1.5 ml tubes (Sarstedt)

Procedure

Note: The procedure was established for cell numbers obtained from small 25 cm2 cell culture flasks. Initially, 1.5 million adherent cells were seeded. After transfection/infection and subsequent cultivation the cells reached 80-100% confluence at day of cell harvest (three days later).

  1. Concentration of viral particles
    1. Centrifuge supernatants of adherent, transfected or infected cells at 300 x g and 4 °C for 5 min to pellet all remaining cellular material. When suspension cells are used, pellet the cells by this centrifugation step. For 25 cm2 flasks 5 ml medium is used during transfection and cultivation. In general, a larger volume of medium can be used as long as the capacity of the ultracentrifugation tubes (see I-8 in materials and step 1-d of the procedure) including the sucrose solution is not exceeded.
    2. Add fresh medium to the transfected or infected cells and place them in the incubator until cytoplasmic RNA isolation.
    3. Filter cell-free supernatants through a 0.45 μm filter.
    4. Ultracentrifuge pre-cleared supernatants through a 30% sucrose cushion. Add 2.5 ml sucrose solution to the tube and carefully overlay with the 5 ml supernatant without disrupting the sucrose pellet. The tubes have to be filled up with serum-free DMEM until 0.5-1 cm below the edge of the tube. This is important to stabilize the tubes during ultracentrifugation. The final volume is determined by precise balancing the tubes in buckets which are inserted opposite each other in the same rotor. Centrifuge at 150,000 x g for 2 h at 4 °C.
  2. Harvest cells
    1. Adherent cells: Discard the freshly added cellular supernatant, wash cells once with PBS (1x; RT) and detach cells with trypsin. Stop trypsin treatment by resuspending cells in serum-containing medium. Pellet cells at 300 x g and 4 °C for 10 min and resuspend them in cold PBS (4 °C).
    2. Suspension cells: Centrifuge cells at 300 x g and 4 °C for 10 min, discard freshly added cellular supernatant and resuspend cells in cold PBS (4 °C).
    3. Pellet cells at 300 x g and 4 °C for 5 min and discard the supernatant. Remove all remaining PBS and loosen the cells by flipping the tube a few times.
    4. During centrifugation steps: Prepare working solutions of RLN and RLT buffers. Prepare 600 μl aliquots of RLT buffer in 1.5 ml tubes. Store both buffers on ice.
  3. Cell fractionation
    1. Resuspend cells carefully in 175 μl ice cold RLN buffer by shortly pipetting them up and down with a wide bored 1,000 μl pipette. Extensive pipetting may result in total cell lysis and should be prevented.
    2. Incubate exactly 5 min on ice to lyse the plasma membrane.
    3. Pellet nuclei by centrifugation at 300 x g for 2 min at 4 °C in a pre-cooled centrifuge.
    4. Transfer the supernatant representing the cytoplasmic fraction to new 1.5 ml tubes with a 200 μl pipette. Be sure not to contaminate the supernatant with the nuclear pellet.
    5. Centrifuge the cytoplasmic fraction at 13,000 x g at 4 °C for 3 min in a pre-cooled centrifuge to pellet all remaining nuclear contaminants.
    6. Transfer supernatant to already prepared 600 μl RLT aliquots (step 2-c) and store on ice.
    7. Optional: Extraction of the nuclear fraction
      1. Resuspend nuclei pellet in 900 μl cold PBS (4 °C) and centrifuge for 5 min at 300 x g and 4 °C in a pre-cooled centrifuge.
      2. Discard PBS, add 600 μl icecold RLT buffer and mix by vortexing. Cell debris will not resolve completely in the RLT buffer.
      3. Load the solution containing the debris onto QIAshredder Spin Columns and centrifuge 2 min at 13,000 x g. Collect the flow through as nuclear fraction.
  4. Cytoplasmic/Nuclear RNA isolation
    1. Vortex nuclear or cytoplasmic fractions in RLT and spin for 5 sec at 13,000 x g and RT.
    2. Add 430 μl ethanol (> 99.8%) and mix by pipetting up and down.
    3. Isolate RNA with RNeasy Mini Kit according to manufacturer’s instructions.
    4. Elute RNA with 47 μl RNase-free H2O (this will result in an elution volume of 45 μl).
    5. Perform DNase digestion with the TURBO DNase-free Kit as follows: Add 5 μl 10x TURBO-DNase buffer and 1 μl or 2 μl TURBO DNase to cytoplasmic or nuclear eluates, respectively. Mix gently and incubate 2-4 h at 37 °C.
    6. Inactivate DNase by adding 5 μl inactivation reagent (included in TURBO DNA-free Kit) per 1 μl DNase used, incubate for 5 min at RT while keeping the reagent resuspended.
    7. Centrifuge 1 min at 11,000 rpm and RT and transfer the supernatant into a fresh tube.
    8. Measure RNA concentration using an RNA-specific dye (e.g. Quant-iT RNA Assay Kit), adjust RNA concentrations with nuclease-free water to e.g. 0.05-0.5 μg/μl and store samples at -80 °C.
  5. Isolation of particle-associated RNA
    1. Carefully remove the supernatant from ultracentrifuged samples from step 1-d and dry the tube walls with a clean wipe.
    2. Resuspend (invisible) pellet in 150 μl PBS by pipetting up and down (Optional: Shake tubes 1 h on ice in advance. This step allows a 1 h interruption of the procedure without any loss of assay performance).
    3. Take 10 μl of concentrated, resuspended particles and store at -20 °C for p24-specific ELISA (HIV-1 DIY p24 sandwich ELISA Kit Protocol 2).
      Note: For analyzing the encapsidation efficiency virion-associated RNA copy numbers need to be normalized to the amount of particles in the supernatant. (Reduced/increased levels of particle-associated RNA can sometimes be attributed to reduced/increased particle levels.) Therefore, determination of p24 levels in the supernatant is important.
    4. Add 560 μl AVL buffer containing carrier RNA (both included in QIAmp Viral RNA Kit) to the samples, vortex and incubate 10 min at RT (crystals appear in the AVL buffer containing carrier RNA when stored at 4 °C. Resolve those precipitations by vigorous shaking for max. 2 min at 50 °C and let the buffer cool down to RT before usage).
    5. Add 560 μl ethanol (> 99.8%) to the samples and vortex vigorously for 15 sec.
    6. Isolate RNA using QIAmp Viral RNA Kit according to the manufacturer’s instructions.
    7. Elute virus-associated RNA with 47 μl RNase-free H2O.
    8. Perform DNase digestion as described in steps 4-e~4-g. An amount of 1 μl DNase is sufficient for particle-RNA samples.
    9. Store samples at -80 °C until further use. Measurement of RNA concentration is not necessary in this case, because the total RNA amount is low and the samples contain unspecific carrier RNA (present in the AVL buffer).
  6. RT-qPCR
    1. Set up RT-qPCR reaction using QuantiTect Probe RT-PCR Kit as follows:
      PCR mix (2x, contains Taq, dNTPs)   
      10 μl                                                                   
      Reverse Transcriptase (RT) enzyme
      0.2 μl
      Primer sense (2.5 μM)
      1 μl
      Primer antisense (2.5 μM)
      1 μl
      SYBR Green (0.1x)
      1 μl
      Isolated RNA 0.5 μg cytoplasmic or 0.05 μg nuclear RNA; 5 μl particle-associated RNA; 5 μl RNA standards ranging from 2 x 101-1 x 109 RNA copies/μl
      Add nuclease-free water to 20 μl.
      Prepare additional samples omitting the RT enzyme in order to detect amplification of (genomic or transfected) DNA sequences.
      Primers used for HIV-1 specific RT-qPCR are used according to the Amplicor HIV-1 Monitor Test (Michael et al., 1999).
      Sense: SK145s (5’-AGT GGG GGG ACA TCA AGC AGC CAT GCA AAT-3’)
      Antisense: SKCC1Bas (5’-TAC TAG TAG TTC CTG CTA TGT CAC TTC C-3’)
    2. RNA standards identical in sequence to the detected HIV RNA segment for absolute quantification of cytoplasmic and particle-associated RNA copy numbers were produced with the AmpliScribe-T7/Sp6 High Yield Transcription Kit (Epicentre). The amplicon obtained with primer pair SK145s/SKCC1Bas after RT-(q)PCR was subcloned into the pCRII-TOPO plasmid with the TOPO TA Cloning Kit. An amplicon containing either the T7 or the SP6 promoter was used as template for the in vitro transcription reaction according to the manufacturer’s instructions. Template DNA was removed by digestion with the MBU DNase from the in vitro transcription kit and in addition by two treatments with the TURBO DNA-free kit. RNA concentrations were measured and adjusted to 2 x 101 to 1 x 109 RNA copies per μl.
    3. Using QuantiTect Probe RT-PCR Kit allows the RT reaction and PCR amplification to happen in one tube without interruption.
      RT-qPCR is performed in a Rotor-Gene Q system with the following settings:
      RT step
      50 °C                                    
      20 min                                 
      Initial denaturation
      95 °C
      15 min
      Denaturation
      95 °C
      10 sec
      Annealing 65 °C
      65 °C
      60 sec
      Elongation and fluorescence
      72 °C
      30 sec
      Detection 1


      Fluorescence detection 2
      78 °C
      15 sec
      Steps( iii-vi) should be repeated in a total of 45 cycles.
    4. After the last PCR cycle the following melting curve measurement is performed:
      Ranging from 50 °C to 99 °C with 1 °C/step, 90 sec equilibration for the first step after that 4 s/step. Since amplification is monitored by SYBRGreen in this RT-qPCR, melting curve analyses are important to verify amplification of specific DNA fragments. Furthermore, regular agarose gel electrophoresis analyses should be included to verify amplification of DNA fragments of the expected size.
    5. Analyze RNA copy numbers with Rotor-Gene Q software using values for fluorescence measurement 2 only. In contrast to measurement 1 fluorescence signals from primer dimers should be strongly reduced at 78 °C. Please note that the exact temperature for the second fluorescence measurement may vary between different Rotor-Gene devices and therefore needs to be optimized.
    6. Note: The primers and settings described here are designed for the detection of HIV-1 unspliced, genomic RNA. To analyze RNA from other retroviruses the RT-qPCR protocol has to be modified.


Figure 1: Schematic diagram of encapsidation assay workflow. Particle-containing supernatants from infected or transfected cells were ultracentrifuged through a 30% sucrose cushion. RNA isolated from concentrated particles and cytoplasm of the infected/transfected cells was analyzed by RT-qPCR. Encapsidation efficiency was determined as the ratio between HIV-1 copy numbers/ml supernatant and HIV-1 copy numbers/μg cytoplasmic RNA. Depending on the experimental design quantification of p24 levels in the concentrated-particle samples is important to normalize particle-associated RNA levels to the amount of p24/Gag particles in the supernatants.

Recipes

  1. Cell culture medium
    DMEM
    10% (v/v) FCS
    1% (v/v) Penicillin/Streptomycin
  2. RLN buffer
    50 mM Tris-Cl (pH 8.0)
    140 mM NaCl
    1.5 mM MgCl2
    0.5% (v/v) Nonidet P-40
    Filter through a 0.2 μm filter.
    Before use add 1,000 U/ml RNase inhibitor and 1 mM DTT.
  3. RLT buffer (included in the RNeasy Mini Kit)
    Before use add 1% (v/v) beta-Mercaptoethanol
  4. AVL buffer (included in the QIAamp Viral RNA Kit)
    Add 1 ml of AVL buffer to one tube of lyophilized carrier RNA (included in the QIAamp Viral RNA Kit), mix thoroughly and transfer the solution to the AVL buffer bottle. After mixing prepare aliquots and store them at 4 °C

Acknowledgments

This protocol was adapted from our papers: Brandt et al. (2007); Blissenbach et al. (2010); and Grewe et al. (2012). This work was funded by a grant from the German Research Foundation (DFG) to Klaus Überla (Ue45/11-1). Bianca Hoffmann is and Bastian Grewe was supported by a fellowship from the DFG graduate school (GRK 1045). Beside Bianca Hoffmann and Bastian Grewe, Inga Ohs, Maik Blissenbach, Sabine Brandt, Bettina Tippler, Thomas Grunwald, Klaus Überla, Rebecca Konietzny, and Klaus Sure were part of the team which established the methods described.

References

  1. Brandt, S., Blissenbach, M., Grewe, B., Konietzny, R., Grunwald, T. and Uberla, K. (2007). Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation. PLoS Pathog 3(4): e54.
  2. Blissenbach, M., Grewe, B., Hoffmann, B., Brandt, S. and Uberla, K. (2010). Nuclear RNA export and packaging functions of HIV-1 Rev revisited. J Virol 84(13): 6598-6604.
  3. Grewe, B., Hoffmann, B., Ohs, I., Blissenbach, M., Brandt, S., Tippler, B., Grunwald, T. and Uberla, K. (2012). Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag. J Virol 86(6): 2990-3002.
  4. Michael, N. L., Herman, S. A., Kwok, S., Dreyer, K., Wang, J., Christopherson, C., Spadoro, J. P., Young, K. K., Polonis, V., McCutchan, F. E., Carr, J., Mascola, J. R., Jagodzinski, L. L. and Robb, M. L. (1999). Development of calibrated viral load standards for group M subtypes of human immunodeficiency virus type 1 and performance of an improved AMPLICOR HIV-1 MONITOR test with isolates of diverse subtypes. J Clin Microbiol 37(8): 2557-2563.

材料和试剂

我。 细胞收获和超速离心

  1. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:41965-039)
  2. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10108-165)
  3. 磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:14200-067)
  4. 胰蛋白酶(Biochrom)
  5. 青霉素/链霉素(Life Technologies,Gibco )
  6. 0.45μm过滤器(Sarstedt)
  7. 蔗糖(Biomol)
  8. 超速离心管(17ml)(Herolab)

II。 细胞分离和RNA分离

  1. 二硫苏糖醇(DTT)溶液(1M)(AppliChem GmbH,目录号:A3668,0050)
  2. RiboLock核糖核酸酶抑制剂(40U /μl)(Thermo Fisher Scientific,目录号:E00384)
  3. 乙醇> 99.8%(Sigma-Aldrich)
  4. QIAshredder旋转柱(QIAGEN,目录号:79654)
  5. RNeasy Kit(QIAGEN,目录号:74106)
  6. QIAmp病毒RNA试剂盒(QIAGEN,目录号:52906)
  7. TURBO DNA无试剂盒(Life Technologies,Ambion ,目录号:AM1907)
  8. 无核酸酶水(B Braun)
  9. Quant-iT RNA测定试剂盒(Life Technologies,Invitrogen ,目录号:Q-33140)

III。 RT-qPCR

  1. QuantiTect Probe RT-PCR试剂盒(包括逆转录酶)(QIAGEN,目录号:204443)
  2. SYBR Green(Life Technologies,Invitrogen TM)
  3. 引物有义和反义(Biomers)
  4. 分离的RNA
  5. TOPO TA克隆试剂盒(Life Technologies,Invitrogen TM ,目录号:45-0640)
  6. AmpliScribe-T7/Sp6高产量转录试剂盒(Epicentre,目录号:AS2607)
  7. RNA标准品

IV。 其他材料

  1. HIV-1 DIY p24夹心ELISA试剂盒2(Aalto Bio Reagents)

设备

  1. Heraeus Multifuge X3R离心机(Thermo Fisher Scientific)
  2. 离心机5417R(Eppendorf)
  3. Sorvall WX Ultra 100超速离心机(Thermo Fisher Scientific)
  4. Qubit荧光计(Life Technologies,Invitrogen TM ,目录号:Q32857)
  5. Rotor-Gene Q(QIAGEN,目录号:9001620)
  6. 带管0.1ml(QIAGEN)
  7. 15ml管(Sarstedt)
  8. 1.5ml管(Sarstedt)

程序

注意:对于从小25cm 2细胞培养瓶获得的细胞数目建立程序。 最初,接种150万个贴壁细胞。 在转染/感染和随后的培养后,细胞在细胞收获日(三天后)达到80-100%汇合。

  1. 病毒颗粒的浓度
    1. 离心的粘附,转染或感染细胞的上清液在300×g和4℃5分钟,沉淀所有剩余的细胞材料。当使用悬浮细胞时,通过该离心步骤沉淀细胞。对于25cm 2烧瓶,在转染和培养期间使用5ml培养基。通常,可以使用更大体积的培养基,只要不超过包括蔗糖溶液的超速离心管(参见材料中的I-8和步骤1-d)的容量。
    2. 向转染或感染的细胞中加入新鲜培养基,将其置于培养箱中,直到细胞质RNA分离
    3. 通过0.45μm过滤器过滤无细胞上清液
    4. 超速离心机通过30%蔗糖垫预清除上清液。向试管中加入2.5ml蔗糖溶液,小心地用5ml上清液覆盖,不破坏蔗糖沉淀。管必须用无血清DMEM填充,直到管的边缘下0.5-1cm。这对于在超速离心期间稳定管是重要的。最终体积通过精确平衡桶中的管来确定 它们在相同的转子中彼此相对地插入。在4℃下以150,000xg离心2小时。
  2. 收获细胞
    1. 贴壁细胞:弃去刚添加的细胞上清液,用PBS(1x; RT)洗涤细胞一次,并用胰蛋白酶分离细胞。停止胰蛋白酶治疗通过重悬细胞在含血清培养基中。将细胞在300×g和4℃下孵育10分钟,并将其重悬于冷PBS(4℃)中。
    2. 悬浮细胞:在300×g和4℃下离心细胞10分钟,弃去新鲜加入的细胞上清液,并将细胞重悬于冷PBS(4℃)中。
    3. 沉淀细胞在300×g和4℃下5分钟,弃去上清液。取出所有剩余的PBS,并通过翻转管子几次来松开细胞
    4. 离心步骤:准备RLN和RLT缓冲液的工作溶液。准备在1.5毫升管中的RLT缓冲液的600微升等分。将两个缓冲液储存在冰上。
  3. 细胞分级
    1. 重悬细胞仔细在175微升冰冷的RLN缓冲液,短暂吸取他们上下与一个广泛的1,000微升移液器。 大量吸液可能导致细胞全部溶解,应予以防止
    2. 在冰上孵育恰好5分钟以裂解质膜
    3. 通过在预冷却的离心机中在4℃下以300×g离心2分钟来沉淀颗粒核。
    4. 转移代表细胞质部分的上清液到新的1.5毫升管用200微升吸管。 确保不要用核沉淀物污染上清液
    5. 在预冷却的离心机中在4℃下将细胞质级分在13,000×g离心3分钟,以沉淀所有剩余的核污染物。
    6. 将上清转移至已制备的600μlRLT等分试样(步骤2-c),并储存在冰上
    7. 可选:提取核部分
      1. 在900μl冷PBS(4℃)中重悬细胞核沉淀,并在预冷的离心机中在300×g和4℃下离心5分钟。
      2. 弃去PBS,加入600μl冰冷的RLT缓冲液,并通过涡旋混合。 细胞碎片在RLT缓冲液中不会完全分解。
      3. 将含有碎片的溶液装载到QIAshredder旋转柱上,并以13,000×g离心2分钟。 收集流量作为核分数。
  4. 细胞质/核RNA分离
    1. 在RLT中涡旋核或细胞质级分,并在13,000×g 和RT下旋转5秒。
    2. 加入430μl乙醇(> 99.8%),并通过上下吹吸混合
    3. 根据制造商的说明,使用RNeasy Mini Kit分离RNA
    4. 用47μl无RNase的H 2 O 2洗脱RNA(这将导致45μl的洗脱体积)。
    5. 使用TURBO DNase-free试剂盒进行DNase消化,如下所示:向细胞质或核洗脱液中分别加入5μl10x TURBO-DNase缓冲液和1μl或2μlTURBO DNase。 轻轻混合并在37℃孵育2-4小时
    6. 通过加入5μl灭活剂(包含在TURBO无DNA试剂盒)每1μlDNA酶灭活DNase,孵育5分钟,在室温下,同时保持试剂重悬。
    7. 在11,000rpm和RT下离心1分钟,并将上清液转移到新管中
    8. 使用RNA特异性染料(例如Quant-iT RNA测定试剂盒)测量RNA浓度,用不含核酸酶的水调节RNA浓度至例如0.05-0.5μg/μl, 存储样品在-80°C
  5. 颗粒相关RNA的分离
    1. 小心地从步骤1-d的超速离心样品中除去上清液,并用干净的擦拭物干燥管壁。
    2. 通过上下吹吸在150μlPBS中重悬(不可见)沉淀(任选:在冰上预先摇动管1小时,此步骤允许1h的程序中断,而没有任何测定性能的损失) 。
    3. 取10μl浓缩的,重悬浮的颗粒,并存储在-20°C p24特异性ELISA(HIV-1 DIY p24夹心ELISA试剂盒协议2)。
      注意:为了分析衣壳化效率,病毒体相关的RNA拷贝数需要相对于上清液中的颗粒量进行标准化。 (颗粒相关RNA的减少/增加的水平有时可归因于减少/增加的颗粒水平。)因此,确定上清液中的p24水平是重要的。
    4. 加入560微升含有载体RNA(均包括在QIAmp病毒RNA试剂盒中)的AVL缓冲液,涡旋并在室温下孵育10分钟(当在4℃下存储时,晶体出现在含有载体RNA的AVL缓冲液中)通过剧烈分解在50℃下摇动最长2分钟,并在使用前将缓冲液冷却至室温)
    5. 向样品中加入560μl乙醇(> 99.8%)并剧烈涡旋15秒
    6. 使用QIAmp病毒RNA试剂盒根据制造商的说明书分离RNA
    7. 用47μl无RNase的H 2 O洗脱病毒相关的RNA
    8. 按步骤4-e〜4-g所述进行DNase消化。 对于颗粒RNA样品,1μlDNA酶的量是足够的
    9. 将样品储存在-80°C直到进一步使用。 在这种情况下不需要测量RNA浓度,因为总RNA量低,并且样品含有非特异性载体RNA(存在于AVL缓冲液中)。
  6. RT-qPCR
    1. 使用QuantiTect Probe RT-PCR Kit设置RT-qPCR反应,如下:
      PCR混合物(2x,含Taq,dNTPs)   
      10μl                           ;                          ;                 
      反转录酶(RT)酶
      0.2μl
      引物检测(2.5μM)
      1微升
      引物反义(2.5μM)
      1微升
      SYBR Green(0.1x)
      1微升
      分离的RNA0.5μg细胞质或0.05μg核RNA; 5微升颗粒相关RNA; 5μlRNA标准品,范围从2×10 1 -1×10 9 RNA拷贝/μl
      加入无核酸酶水至20μl 准备其他样品省略RT酶,以检测(基因组或转染的)DNA序列的扩增 用于HIV-1特异性RT-qPCR的引物根据Amplicor HIV-1监测试验(Michael等人,1999)使用。
      检测:SK145s(5'-AGT GGG GGG ACA TCA AGC AGC CAT GCA AAT-3')
      反义:SKCC1Bas(5'-TAC TAG TAG TTC CTG C​​TA TGT CAC TTC C-3')
    2. 使用AmpliScribe-T7/Sp6高产量转录试剂盒(Epicentre)产生与检测到的HIV RNA片段序列相同的RNA标准物,用于绝对定量胞质和颗粒相关的RNA拷贝数。在RT-(q)PCR后用引物对SK145s/SKCC1Bas获得的扩增子用TOPO TA克隆试剂盒亚克隆到pCRII-TOPO质粒中。根据制造商的说明书,将含有T7或SP6启动子的扩增子用作体外转录反应的模板。通过用来自体外转录试剂盒的MBU DNA酶消化除去模板DNA,另外两个 治疗与TURBO无DNA试剂盒。测量RNA浓度并调整为每μl2×10 6至1×10 9个RNA拷贝。
    3. 使用QuantiTect Probe RT-PCR Kit允许RT反应和PCR扩增在一个管中进行,不间断 RT-qPCR在Rotor-Gene Q系统中进行,具有以下设置:
      RT步骤
      50°C                                     
      20分钟                                  
      初始变性
      95°C
      15分钟
      变性
      95°C
      10秒
      退火65°C
      65℃
      60秒
      伸长和荧光
      72℃
      30秒
      检测1


      荧光检测2
      78℃
      15秒
      步骤(iii-vi)应该重复总共45个循环。
    4. 在最后一个PCR循环后,进行以下熔解曲线测量:
      在50℃至99℃范围内以1℃/步进行,在4秒/步之后的第一步平衡90秒。 由于在该RT-qPCR中由SYBRGreen监测扩增,熔解曲线分析对于验证特定DNA片段的扩增是重要的。 此外,应包括常规琼脂糖凝胶电泳分析以验证预期大小的DNA片段的扩增
    5. 用Rotor-Gene Q软件仅使用荧光测量值2分析RNA拷贝数。与测量1相反,来自引物二聚体的荧光信号应当在78℃强烈降低。请注意,第二次荧光测量的确切温度可能因不同的Rotor-Gene设备而异,因此需要优化。
    6. 注意:这里描述的引物和设置是为检测HIV-1未剪接的基因组RNA而设计的。为了分析来自其他逆转录病毒的RNA,必须修改RT-qPCR方案。


图1:壳体化测定工作流程示意图。 包含粒子  将来自感染或转染细胞的上清液超速离心 通过30%蔗糖垫。从浓缩颗粒分离的RNA 并通过RT-qPCR分析感染/转染的细胞的细胞质。 包封效率确定为HIV-1拷贝之间的比率   数量/ml上清液和HIV-1拷贝数/μg细胞质RNA。 取决于p24水平的实验设计定量   浓缩颗粒样品对归一化很重要 颗粒相关的RNA水平与p24/Gag颗粒的量   上清液。

食谱

  1. 细胞培养基
    DMEM
    10%(v/v)FCS
    1%(v/v)青霉素/链霉素
  2. RLN缓冲区
    50mM Tris-Cl(pH8.0) 140mM NaCl 1.5mM MgCl 2·h/v 0.5%(v/v)Nonidet P-40
    通过0.2μm过滤器过滤 使用前加入1,000U/ml RNA酶抑制剂和1mM DTT
  3. RLT缓冲区(包含在RNeasy Mini Kit中)
    使用前加入1%(v/v)β-巯基乙醇
  4. AVL缓冲液(包含在QIAamp病毒RNA试剂盒中) 加入1毫升AVL缓冲液到一个管的冻干载体RNA(包括在QIAamp病毒RNA试剂盒),彻底混合,并将溶液转移到AVL缓冲瓶。 混合后,准备等分试样,并在4℃下保存

致谢

该协议改编自我们的论文:Brandt等人(2007); Blissenbach等人(2010);和Grewe等人(2012)。这项工作是由德国研究基金会(DFG)授予KlausÜberla(Ue45/11-1)资助的。 Bianca Hoffmann和Bastian Grewe得到了DFG研究生院(GRK 1045)的支持。在Bianca Hoffmann和Bastian Grewe,Inga Ohs,Maik Blissenbach,Sabine Brandt,Bettina Tippler,Thomas Grunwald,KlausÜberla,Rebecca Konietzny和Klaus Sure之后,他们都是建立所述方法的团队的一部分。  

参考文献

  1. Brandt,S.,Blissenbach,M.,Grewe,B.,Konietzny,R.,Grunwald,T.and Uberla,K。 人类和猴免疫缺陷病毒的Rev蛋白增强RNA包被。 3(4):e54。
  2. Blissenbach,M.,Grewe,B.,Hoffmann,B.,Brandt,S.and Uberla,K。(2010)。 重新审视HIV-1 Rev的核RNA出口和包装功能。 J Virol 84(13):6598-6604。
  3. Grewe,B.,Hoffmann,B.,Ohs,I.,Blissenbach,M.,Brandt,S.,Tippler,B.,Grunwald,T.and Uberla,K。 1型人类免疫缺陷病毒基因组RNA的细胞质利用不依赖于与gag的核相互作用。 a>。 J Virol 86(6):2990-3002。
  4. Michael,NL,Herman,SA,Kwok,S.,Dreyer,K.,Wang,J.,Christopherson,C.,Spadoro,JP,Young,KK,Polonis,V.,McCutchan,FE,Carr, Mascola,JR,Jagodzinski,LLand Robb,ML(1999)。 针对M型人类免疫缺陷病毒1型亚型的校正病毒载量标准的开发和改进的 AMPLICOR HIV-1 MONITOR测试与不同亚型的分离株。 J Clin Microbiol 37(8):2557-2563
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How to cite this protocol: Hoffmann, B. and Grewe, B. (2013). Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR. Bio-protocol 3(8): e684. DOI: 10.21769/BioProtoc.684; Full Text



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