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Quantification of Retro- and Lentiviral Reverse Transcriptase Activity by Real-time PCR
实时定量PCR(qPCR)量化分析逆转录和慢病毒逆转录酶活性

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Abstract

Quantification of retroviral reverse transcriptase activity in retrovirus containing supernatant by quantitative reverse transcription PCR as a method for titration of HIV, lenti- and retroviral vectors is described here.. The procedure was optimized for use with LightCycler 480 (Roche, Vilvoorde, Belgium) and ABI 7300 real-time PCR system (reagents and procedures that are system specific will be marked accordingly in the protocol).

Materials and Reagents

  1. MS2 RNA, aliquoted per 30 μl, storage -20 °C (F. Hoffmann-La Roche, catalog number: 10165948001 )
  2. Ribolock RNase inhibitor 40 U/μl, aliquoted per 10 μl, storage -20 °C (Fermentas, catalog number: EO0381 )
  3. HIV Reverse Transcriptase (Life Technologies, Ambion®, catalog number: AM2045 )
  4. Primers (directed against MS2 cDNA), storage -20 °C, (Eurogentec, Seraing, Belgium):
    FWD: 5'TCCTGCTCAACTTCCTGTCGAG3'
    REV: 5'CACAGGTCAAACCTCCTAGGAATG3'
  5. Nuclease-free water, further referred to as H2O (Life Technologies, catalog number: AM9939 )
  6. [Light Cycler 480]: LightCycler 480 SybrGreen I Master: Protect against light with aluminium foil (F. Hoffmann-La Roche, catalog number: 04707516001)
    OR [ABI 7300]: Eurogentec qPCR core kit for SYBR Green I (Eurogentec, catalog number: RT-QP73-05 )
  7. Tris (Sigma-Aldrich, catalog number: 154563-500G )
  8. Ultrapure water (MilliQ water filtered with Milli-Q Gradient System) (Merck Millipore, Overijse, Belgium)
  9. KCl (UCB, catalog number: 1592 )
  10. Glycerol (Merck Millipore, catalog number: 1.04094.1000 )
  11. Triton-X100 (MP Biomedicals, catalog number: 807423 )
  12. 2x Lysis buffer (Homemade), storage -20 °C (see Recipes)
  13. Eurogentec qPCR core kit for SYBR Green I (see Recipes)

Equipment

  1. Tissue Culture Plate 96W U-bottom (BD Biosciences, Falcon®, catalog number: 353077 or similar)
  2. [Light Cycler 480]: LightCycler 480 Sealing foil (F. Hoffmann-La Roche, catalog number: 04729757001 ) OR [ABI 7300]: Optical adhesive film (Applied Biosystems, catalog number: 4311971 )
  3. [Light Cycler 480]: LightCycler 480 Multiwell plate 384 (F. Hoffmann-La Roche, catalog number: 04729749001 ) OR [ABI 7300]: MicroAmp Optical 96-well reaction plate with barcode (Applied Biosystems, catalog number: 4306737 )
  4. MicroAmp adhesive film applicator (Applied Biosystems, catalog number: 4333183 )
  5. Aluminium foil
  6. Table top centrifuge

Procedure

Overview of the procedure:

  1. Preparations (see I): Make the lysis buffer / RNase inhibitor mix and qRT-PCR reaction mix.
  2. Lysis of the viral samples (see III.): Addition of lysis buffer to the viral supernatant to release reverse transcriptase.
  3. qRT-PCR (see III): Addition of qRT-PCR reaction mix to the lysed samples, followed by the reverse transcription and quantitative PCR reaction.
  4. Analysis (see IV): Calculation of the reverse trancritpase activity of the samples based on the obtained standard curve.

I.   Preparations:

  1. Calculate the amount of samples that will be included in the RT assay (x samples).
    Always include:
    1. Standard curve (SC) (containing 7 dilutions of viral supernatant or 7 dilutions of recombinant HIV reverse transcriptase + 1 medium control = 8 samples).
    2. 3 control viral supernatant (C ) (= 3 samples)
    3. your samples (S)
  2. Make a mixture of 2x lysis buffer and RNase inhibitor (40 U/μl) (see Table 1):
    Table 1. Required Lysis buffer/RNase inhibitor

    Reagent per reaction
    (1 sample)
    Reagent for all reactions
    ((x *1.25) samples)
    2x lysis buffer
    5 μl
    5*(x*1.25) μl
    RNase inhibitor (40 U/μl)
    0.1 μl
    0.1* (x*1.25) μl
    Notes:
    1) lysis buffer is very viscious. Therefore always make 25% extra and do not vortex to avoid formation of bubbles
    .
    2) keep RNase inhibitor on ice.
  3. Calculate the amount of samples that you will measure by qRT-PCR (n samples)
    Always include:
    1. Each sample in duplicate (= x*2 samples)
    2. H2O twice (= 2 samples) (negative control of the qPCR)
  4. Make the qRT-PCR reaction mix (see Table 2a [Light Cycler 480] or Table 2b [ABI 7300].):
    Notes:
    1) Make 10% extra
    .
    2) Keep LC 480 Sybr Green I mix, MS2 RNA and RNase inhibitor on ice.
    3) Do not vortex LC480 Sybr Green I mix.
    4) RNase inhibitor has to be diluted 10x in H2O (final concentration: 4 U/μl).
    Table 2a. qRT-PCR reaction mix for use on LightCycler 480 (384 well plates)


    Reagent per reaction (1 sample)
    Reagent for all reactions ((n *1.1) samples)
    LC 480 Sybr Green I (2x)
    10 μl
    10*(n*1.1) μl
    Fwd primer (100 μM)
    0.1 μl
    0.1* (n*1.1) μl
    Reverse primer (100 μM)
    0.1 μl
    0.1* (n*1.1) μl
    MS2 RNA
    0.1 μl
    0.1* (n*1.1) μl
    RNase inhibitor (10x diluted in H2O, final concentration: 4 U/μl)
    0.1 μl
    0.1* (n*1.1) μl

    Table 2b. qRT-PCR reaction mix for use on ABI 7300 real-time PCR system (96 well plates)


    1 sample
    (n *1.1) samples
    Eurogentec qPCR core kit for SYBR Green I
    10.6 μl
    10*(n*1.1) μl
    Fwd primer (100 μM)
    0.1 μl
    0.1* (n*1.1) μl
    Reverse primer (100 μM)
    0.1 μl
    0.1* (n*1.1) μl
    MS2 RNA
    0.1 μl
    0.1* (n*1.1) μl
    RNase inhibitor (10x diluted in H2O, final concentration: 4 U/μl)
    0.1 μl
    0.1* (n*1.1) μl

II.  Lysis of the viral samples

  1. Add 5 μl of the viral supernatant or standard curve per well of a 96W U bottom plate.
  2. Add 5 μl of Lysis buffer/RNase mix to each well and mix.
  3. Incubate 10 min at room temperature (RT).
  4. Add 90 μl nuclease-free water to each well and mix.
  5. Spin plate for 3 min at 1,600 x g at RT in table top centrifuge.
  6. Keep the plate on ice until transfer to the 384W [LightCycler 480] OR 96W [ABI 7300] qPCR plate.

III. qRT-PCR

  1. Take a cooling element out of the -20 °C and cover the cooling element with aluminium foil. Put the 384W [LightCycler 480] OR 96W [ABI 7300] plate on the aluminium foil during the filling of the plate to prevent evaporation.
  2. Put 10.4 μl [LightCycler 480] OR 11 μl [ABI 7300] of qRT-PCR reaction mix in each well of 384W [LightCycler 480] OR 96W [ABI 7300] plate.
  3. Resuspend all the viral samples in the lysate plate.
  4. Add 9.6 μl [LightCycler 480] OR 9 μl [ABI 7300] of sample to each well and resuspend.
  5. Seal the plate using Sealing foil [LightCycler 480] OR Optical Adhesive film [ABI 7300], by removing the protective layer and press it to the plate using the applicator.
  6. Spin the plate at 1,600 x g for 3 min at RT in table top centrifuge.
  7. Run reaction:
    1. Light Cycler 480:
      Detection format: Sybr Green/HRM dye
      Reaction volume: 20 μl
      Program:
      Step 1: Reverse transcription (rt): 42 °C for 20 min.
      Step 2: Pre-incubation: 95 °C for 5 min.
      Step 3: Amplification: 40 cycles of
                  95 °C for 5 sec
                  60 °C for 5 sec + detection
                  72 °C for 15 sec
      Step 4: Melting curve
      Step 5: Cooling
    2. Run on the ABI Prism 7300:
      Reaction volume: 20 μl
      Program:
      Step 1: Reverse transcription (rt): 42 °C for 20 min.
      Step 2: Pre-incubation: 95 °C for 2 min.
      Step 3: Amplification: 40 cycles of
                  95 °C for 5 sec
                  60 °C for 30 sec + detection
                  72 °C for 15 sec
      Step 4: Melting curve

IV. Analysis

  1. Export the obtained Cq (Cycle of quantification) values from the LC480 or ABI Prism 7300.
  2. Calculate for each sample the average Cq value of the duplicate measurements.
  3. Make the standard curve: plot the average Cq value of SC1-SC7 versus the logarithm of the RT activity (expressed as mU RT/ml), determine the trendline and the formula expressing the correlation between the Cq values and the logarithm of the RT activity.
  4. Use the obtained formula to calculate the absolute RT value of the samples.

Notes

Control samples:

  1. Standard curve:
    For absolute quantification of retroviral RT activity one can measure a serial dilution with known concentration of recombinant reverse transcriptase in parallel with the samples of interest (see a). Alternatively, a standard curve can be made by using serial dilution of a retro- or lentiviral supernatant of choice. In the latter case, it necessary to determine the RT activity of this supernatant in a first experiment by running a recombinant reverse transcriptase standard curve in parallel. For later experiment the serial dilution of the retroviral supernatant can be used as a standard curve (see b).
    1. Use recombinant HIV Reverse transcriptase as standard curve
      SC1 = solution of 200 mU/μl HIV Reverse Transcriptase. This is 1/50 dilution of the stock solution (10 U/μl)
      SC2-SC7 are made by serial 1/10 dilution of SC1 in cell culture medium (preferably the same medium in which retro- and lentiviruses were produced)
      Sample SC8 = cell culture medium (negative control)
    2. Use high titer retroviral supernatant as a standard curve
      SC1 = high titer retro- or lentiviral supernatant of your choice.
      This sample can be any viral supernatant that is more concentrated than the samples being analysed. As mentioned above, the RT activity of this sample should be determined in an initial experiment by running a recombinant reverse transcriptase standard curve (see a) in parallel.
      SC2-SC7 are made by serial 1/10 dilution of SC1 in cell culture medium (preferably the same medium in which retro- and lentiviruses are produced)
      Sample SC8 = cell culture medium
      All samples are aliquoted per 8.5 μl in PCR strips. For each assay a new strip will be used.
  2. Control samples C1, C2, C3:
    3 retroviral supernatants of your choice that will be measured in each RT assay for quality control: The obtained RT activity of the control samples should be similar over different RT assays and offers a control for interrun variation.

Recipes

  1. 1 M Tris-HCl solution
    12.1 g Tris in 50 ml ultrapure water-adjust pH to 7.4 with HCl
    Adjust total volume to 100 ml with MilliQ water
  2. 2x Lysis buffer (storage -20 °C)
    0.25% Triton X-100, 50 mM KCl, 100 mM TrisHCl, pH 7.4, 40% glycerol
    Add 50 ml of 1 M TRIS-HCl solution to a 500 ml bottle
    Add 1.86 g KCl to the 500 ml bottle
    Add 200 ml glycerol
    Add 1.25 ml of Triton-X100
    Mix the solution very well until everything is dissolved
    Add MilliQ water up to a final volume of 500 ml
    Aliquot 40 ml in 1.5 ml screw cap microtube
    Aliquot the remainder in 50 ml tubes
  3. Eurogentec qPCR core kit for SYBR Green I (store aliquots at -20 °C)
    Composition of 10.6 μl:
    2 μl 10x reaction buffer
    1.4 μl of 50 mM MgCl2
    0.8 μl of 5 mM dNTP mix
    0.1 μl of HotGoldS Tar Taq polymerase
    0.6 μl SYBR Green I
    5.7 μl of nuclease-free water

Acknowledgments

The presented assay is an adapted version of the SG-PERT assay described before in Pizzato et al. (2009). This work was supported by SBO CellCoVir grant from the agency for Innovation by Science and Technology (IWT) Flanders, Belgium; HIV-STOP Interuniversity Attraction Poles program of Belgian Science Policy, European Union FP7 Health-2007-2.3.2-1 Collaborative Project iNEF, Ghent University grant BOF11/GOA/013 and grants from the Research Foundation – Flanders (FWO).

References

  1. Pizzato, M., Erlwein, O., Bonsall, D., Kaye, S., Muir, D. and McClure, M. O. (2009). A one-step SYBR Green I-based product-enhanced reverse transcriptase assay for the quantitation of retroviruses in cell culture supernatants. J Virol Methods 156(1-2): 1-7.
  2. Vermeire, J., Naessens, E., Vanderstraeten, H., Landi, A., Iannucci, V., Van Nuffel, A., Taghon, T., Pizzato, M. and Verhasselt, B. (2012). Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors. PLoS One 7(12): e50859.

简介

本文描述了通过定量逆转录PCR作为滴定HIV,慢病毒和逆转录病毒载体的逆转录病毒逆转录病毒逆转录酶活性的逆转录病毒逆转录酶活性的定量。将该程序优化用于LightCycler 480(Roche,Vilvoorde,Belgium) ABI 7300实时PCR系统(系统特异性的试剂和程序将在协议中相应地标记)

材料和试剂

  1. MS2 RNA,每30μl分装,-20℃保存(F.Hoffmann-La Roche,目录号:10165948001)
  2. Ribolock RNA酶抑制剂40U /μl,每10μl等分,保存-20℃(Fermentas,目录号:EO0381)
  3. HIV逆转录酶(Life Technologies,Ambion ,目录号:AM2045)
  4. 引物(针对MS2 cDNA),储存-20℃,(Eurogentec,Seraing,Belgium):
    FWD:5'TCCTGCTCAACTTCCTGTCGAG3'
    REV:5'CACAGGTCAAACCTCCTAGGAATG3'
  5. 无核酸酶水,进一步称为H 2 O(Life Technologies,目录号:AM9939)
  6. [Light Cycler 480]:LightCycler 480 SybrGreen I Master:用铝箔防止光照(F.Hoffmann-La Roche,目录号:04707516001)
    OR [ABI 7300]:用于SYBR Green I(Eurogentec,目录号:RT-QP73-05)的Eurogentec qPCR核心试剂盒
  7. Tris(Sigma-Aldrich,目录号:154563-500G)
  8. 超纯水(MilliQ水,用Milli-Q梯度系统过滤)(Merck Millipore,Overijse,Belgium)
  9. KCl(UCB,目录号:1592)
  10. 甘油(Merck Millipore,目录号:1.04094.1000)
  11. Triton-X100(MP Biomedicals,目录号:807423)
  12. 2x裂解缓冲液(自制),储存-20℃(见配方)
  13. Eurogentec qPCR核心套件用于SYBR Green I(参见配方)

设备

  1. 组织培养板96W U-底(BD Biosciences,Falcon ,目录号:353077或类似物)
  2. [Light Cycler 480]:LightCycler 480密封箔(F.Hoffmann-La Roche,目录号:04729757001)或[ABI 7300]:光学粘合剂膜(Applied Biosystems,目录号:4311971)
  3. [Light Cycler 480]:LightCycler 480多孔板384(F.Hoffmann-La Roche,目录号:04729749001)或[ABI 7300]:具有条形码的MicroAmp光学96孔反应板(Applied Biosystems,目录号:4306737) />
  4. MicroAmp粘性膜涂布器(Applied Biosystems,目录号:4333183)
  5. 铝箔
  6. 台式离心机

程序

程序概述:

  1. 制备(见I):制备裂解缓冲液/RNA酶抑制剂混合物和qRT-PCR反应混合物。
  2. 病毒样品的裂解(见III):向病毒上清液中加入裂解缓冲液以释放逆转录酶。
  3. qRT-PCR(见III):向裂解的样品中加入qRT-PCR反应混合物,随后进行逆转录和定量PCR反应。
  4. 分析(参见IV):基于获得的标准曲线计算样品的反向透明酶活性。

I.   准备:

  1. 计算RT测定中包含的样品量(x个样品)。
    始终包括:
    1. 标准曲线(SC)(含有7个病毒上清液稀释液或7个重组HIV逆转录酶+ 1个培养基对照的稀释液= 8个样品)。
    2. 3对照病毒上清液(C)(= 3个样品)
    3. 您的样品(S)
  2. 制备2x裂解缓冲液和RNA酶抑制剂(40U /μl)的混合物(见表1):
    表1.必需的裂解缓冲液/RNA酶抑制剂

    试剂每次反应
    (1个样本)
    所有反应的试剂
    ((x * 1.25)个样本)
    2x裂解缓冲液
    5微升
    5 *(x * 1.25)μl
    RNase抑制剂(40 U /μl)
    0.1μl
    0.1 *(x * 1.25)μl
    注意:
    1)裂解缓冲液非常粘稠。 因此,请始终提供25%的额外费用,避免形成气泡。 。
    2)将核糖核酸酶抑制剂保持在冰上 。
  3. 计算将通过qRT-PCR(n个样品)测量的样品量
    始终包括:
    1. 每个样品重复(= x * 2个样品)
    2. H 2 O 2(= 2个样品)(qPCR的阴性对照)
  4. 制作qRT-PCR反应混合物(见表2a [Light Cycler 480]或表2b [ABI 7300]。):
    注意:
    1)多赚10%。
    2)保持LC 480 Sybr Green I在冰上混合,MS2 RNA和RNA酶抑制剂。
    3)不要涡旋LC480 Sybr Green I mix 。 4)RNase抑制剂必须在H O(终浓度:4U /μl)中稀释10倍。 。
    表2a。 用于LightCycler 480(384孔板)的qRT-PCR反应混合物

    试剂每次反应(1个样品)
    所有反应的试剂((n * 1.1)样品)
    LC 480 Sybr Green I(2x)
    10微升
    10 *(n * 1.1)μl
    Fwd引物(100μM)
    0.1μl
    0.1 *(n * 1.1)μl
    反向引物(100μM)
    0.1μl
    0.1 *(n * 1.1)μl
    MS2 RNA
    0.1μl
    0.1 *(n * 1.1)μl
    RNase抑制剂(在H 2 O中稀释10倍,终浓度:4U /μl)
    0.1μl
    0.1 *(n * 1.1)μl

    表2b。 用于ABI 7300实时PCR系统(96孔板)的qRT-PCR反应混合物

    1个样品
    (n * 1.1)个样品
    Eurogentec qPCR核心套件,用于SYBR Green I
    10.6微升
    10 *(n * 1.1)μl
    Fwd引物(100μM)
    0.1μl
    0.1 *(n * 1.1)μl
    反向引物(100μM)
    0.1μl
    0.1 *(n * 1.1)μl
    MS2 RNA
    0.1μl
    0.1 *(n * 1.1)μl
    RNase抑制剂(在H 2 O中稀释10倍,终浓度:4U /μl)
    0.1μl
    0.1 *(n * 1.1)μl

II。  病毒样品的裂解

  1. 每个96W U底板每孔加入5μl病毒上清液或标准曲线
  2. 向每个孔中加入5μl裂解缓冲液/RNase混合物并混合
  3. 在室温(RT)下孵育10分钟。
  4. 向每个孔中加入90μl无核酸酶的水并混合。
  5. 在台式离心机中在室温下以1,600×g /分钟旋转板3分钟
  6. 将板保持在冰上,直到转移到384W [LightCycler 480] OR 96W [ABI 7300] qPCR板。

III。 qRT-PCR

  1. 将冷却元件从-20°C取出,并用铝箔覆盖冷却元件。 在填充板期间将384W [LightCycler 480]或96W [ABI 7300]板放在铝箔上,以防止蒸发。
  2. 在384W [LightCycler 480] OR 96W [ABI 7300]板的每个孔中加入10.4μl[LightCycler 480]或11μl[ABI 7300] qRT-PCR反应混合物。
  3. 重悬细胞裂解物板中的所有病毒样品。
  4. 向每个孔中加入9.6μl[LightCycler 480]或9μl[ABI 7300]样品,并重悬。
  5. 使用密封箔[LightCycler 480]或光学粘合膜[ABI 7300],通过去除保护层并使用涂布器将其压到板上来密封板。
  6. 在台式离心机中在室温下将板以1,600×g离心3分钟。
  7. 运行反应:
    1. 光循环仪480:
      检测格式:Sybr Green/HRM染料
      反应体积:20μl
      计划:
      步骤1:反转录(rt):42℃20分钟 步骤2:预温育:95℃5分钟 步骤3:扩增:40个循环的
                   95°C 5秒
                   60°C 5秒+检测
                   72°C 15秒
      第4步:熔解曲线
      第5步:冷却
    2. 在ABI Prism 7300上运行:
      反应体积:20μl
      计划:
      步骤1:反转录(rt):42℃20分钟 步骤2:预温育:95℃2分钟 步骤3:扩增:40个循环的
                   95°C 5秒
                   60°C,30秒+检测
                   72°C 15秒
      第4步:熔解曲线

IV。 分析

  1. 从LC480或ABI Prism 7300导出获得的Cq(定量循环)值。
  2. 计算每个样品的重复测量的平均Cq值
  3. 制作标准曲线:绘制SC1-SC7的平均Cq值与RT活性的对数(表示为mU RT/ml),确定趋势线和表达Cq值与RT活性的对数之间的相关性的公式 。
  4. 使用获得的公式计算样品的绝对RT值

笔记

对照样品:

  1. 标准曲线:
    对于逆转录病毒RT活性的绝对定量,可以用已知浓度的重组逆转录酶与感兴趣的样品平行测量连续稀释(参见a)。或者,可以通过使用选择的逆转录病毒或慢病毒上清液的系列稀释来制备标准曲线。在后一种情况下,必须在第一个实验中通过平行运行重组逆转录酶标准曲线来确定该上清液的RT活性。对于以后的实验,可以使用逆转录病毒上清液的系列稀释作为标准曲线(参见b)
    1. 使用重组HIV逆转录酶作为标准曲线
      SC1 = 200mU /μlHIV逆转录酶的溶液。这是储备溶液的1/50稀释液(10U /μl
      SC2-SC7通过SC1在细胞培养基(优选其中产生逆转录病毒和慢病毒的相同培养基)中连续稀释1/10来制备。
      样品SC8 =细胞培养基(阴性对照)
    2. 使用高滴度逆转录病毒上清液作为标准曲线
      SC1 =您选择的高效价逆转录病毒或慢病毒上清。
      该样品可以是比被分析的样品更浓的任何病毒上清液。如上所述,该样品的RT活性应当在初始实验中通过平行运行重组逆转录酶标准曲线(参见a)来确定。
      SC2-SC7通过SC1在细胞培养基(优选产生逆转录病毒和慢病毒的相同培养基)中的系列1/10稀释制备。
      样品SC8 =细胞培养基
      将所有样品每8.5μl等分到PCR条中。对于每个测定,将使用新的条。
  2. 控制样品C1,C2,C3:
    您选择的3种逆转录病毒上清液将在每个RT测定中进行质量控制:对照样品的RT活性在不同的RT测定中应该是相似的,并且提供了菌株间变异的控制。

食谱

  1. 1M Tris-HCl溶液
    12.1g Tris在50ml超纯水中 - 用HCl调节pH至7.4 用MilliQ水将总体积调整到100 ml
  2. 2×裂解缓冲液(储存-20℃) 0.25%Triton X-100,50mM KCl,100mM TrisHCl,pH7.4,40%甘油 向500 ml瓶中加入50 ml 1 M TRIS-HCl溶液
    向500 ml瓶中加入1.86 g KCl
    加入200ml甘油
    加入1.25ml Triton-X100
    混合溶液很好,直到一切都溶解
    加入MilliQ水至最终体积为500 ml
    等分40毫升在1.5毫升螺旋盖微量管
    将剩余物分装在50ml管中
  3. Eurogentec qPCR核心试剂盒,用于SYBR Green I(在-20°C存储等份) 10.6μl的组成:
    2μl10x反应缓冲液
    1.4μl的50mM MgCl 2·dub 0.8μl5mM dNTP混合物
    0.1μlHotGoldS Tar Taq聚合酶
    0.6μlSYBR Green I
    5.7μl无核酸酶水

致谢

所呈现的测定是先前在Pizzato等人(2009)中描述的SG-PERT测定的适应版本。 这项工作得到了来自比利时佛兰德斯科技创新机构(IWT)的SBO CellCoVir资助; HIV-STOP国际大学吸引力比利时科学政策波兰计划,欧盟FP7健康2007-2.3.2-1合作项目iNEF,根特大学授予BOF11/GOA/013和研究基金会佛兰德斯(FWO)的赠款。

参考文献

  1. Pizzato,M.,Erlwein,O.,Bonsall,D.,Kaye,S.,Muir,D.and McClure,M.O。(2009)。 用于定量逆转录病毒的一步式SYBR Green I产品增强型逆转录酶测定 细胞培养物上清液。病毒学方法 156(1-2):1-7
  2. Vermeire,J.,Naessens,E.,Vanderstraeten,H.,Landi,A.,Iannucci,V.,Van Nuffel,A.,Taghon,T.,Pizzato,M.and Verhasselt, 通过实时PCR作为一种快速,准确的滴定HIV的方法来量化逆转录酶活性, 慢病毒和逆转录病毒载体。 PLoS One 7(12):e50859。
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引用:Vermeire, J. and Verhasselt, B. (2013). Quantification of Retro- and Lentiviral Reverse Transcriptase Activity by Real-time PCR. Bio-protocol 3(11): e780. DOI: 10.21769/BioProtoc.780.
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