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The reverse transcription (RT) reaction is a critical step in HIV-1 life cycle. It is very strongly regulated and the target of several restriction factors (TRIM5α, APOBECs, SAMHD1, etc.). The progress of reverse transcription can be followed by measuring viral DNA by quantitative PCR (qPCR). This method is sensitive enough to allow detection of low amounts of HIV-1 DNA in infected cells and discriminate between several types of reverse transcription intermediates (so called 《early》 and 《late》 RT products, 2 Long Terminal Repeat (LTR) circles, integrated DNA).

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Quantification of HIV-1 DNA
HIV-1 DNA的量化

微生物学 > 微生物遗传学 > DNA > DNA 定量
作者: Ferdinand Roesch
Ferdinand RoeschAffiliation: Institut Pasteur, Unité Virus et Immunité, Département de Virologie, Paris, France
Bio-protocol author page: a313
 and Olivier Schwartz
Olivier SchwartzAffiliation: Institut Pasteur, Unité Virus et Immunité, Département de Virologie, Paris, France
For correspondence: schwartz@pasteur.fr
Bio-protocol author page: a314
Vol 3, Iss 7, 4/5/2013, 4857 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.417

[Abstract] The reverse transcription (RT) reaction is a critical step in HIV-1 life cycle. It is very strongly regulated and the target of several restriction factors (TRIM5α, APOBECs, SAMHD1, etc.). The progress of reverse transcription can be followed by measuring viral DNA by quantitative PCR (qPCR). This method is sensitive enough to allow detection of low amounts of HIV-1 DNA in infected cells and discriminate between several types of reverse transcription intermediates (so called 《early》 and 《late》 RT products, 2 Long Terminal Repeat (LTR) circles, integrated DNA).

[Abstract] 逆转录(RT)反应是HIV-1生命周期中的关键步骤。 它是非常强烈的调节和几个限制因素(TRIM5α,APOBECs,SAMHD1等)的目标。 逆转录的进程可以通过定量PCR(qPCR)测量病毒DNA。 该方法足够灵敏以允许在感染的细胞中检测到少量的HIV-1 DNA,并区分几种类型的逆转录中间体(所谓的"早期"和"晚期"RT产物,2个长末端重复(LTR) 整合DNA)。

Materials and Reagents

  1. Cells of interest, infected with HIV-1
  2. Proteinase K (20 mg/ml) (> 30 U/mg) (Eurobio, catalog number: GEXPRK01-B5 )
  3. AL buffer (QIAEN, catalog number: 19075 )
  4. Phenol-chloroform – isoamyl alcohol (Sigma-Aldrich, catalog number: 77617-100 ml )
  5. Chloroform (Sigma-Aldrich)
  6. Pure water (Life Technologies, Gibco®)
  7. Absolute Ethanol (Sigma-Aldrich)
  8. 3 M sodium acetate (pH 5.0)
  9. DpnI Fast Digest restriction enzyme and reaction buffer (Fermentas, catalog number: FD1704 )
  10. Platinium SYBR Green qPCR Supermix UDG (Life Technologies, InvitrogenTM, catalog number: 11733-046 )
  11. qPCR primers
  12. Standard plasmids for HIV-1 (pNL4-3) and GAPDH (pGAPDH)

Equipment

  1. Centrifuge
  2. DNA-free hood
  3. Speed-vac
  4. Heat-seal machine
  5. qPCR machine 
  6. 96-well qPCR plates (Eppendorf)
  7. Plastic films for qPCR plates (Eppendorf)
  8. Filter tips

Procedure

  1. Collect at least 5 x 105 cells of interest infected with HIV-1, in 1.5 ml Eppendorf tubes and centrifuge for 3 min at 200 x g. Discard supernatant and wash pellets once in 1 ml of sterile PBS. Centrifuge for 3 min at 200 x g and store dried pellets at -80 °C up to a few months.
  2. Open a new bottle of sterile PBS. Resuspend thoroughly the pellets in 180 μl of PBS. Add 20 μl (12 U) of Proteinase K and 200 μl of AL buffer. Vortex vigorously and incubate for 1 h at 56 °C.
  3. Add 400 μl of phenol-chloroform. Vortex vigorously and centrifuge 5 min at 16,200 x g. Collect the upper phase and add 400 μl of chloroform. Vortex vigorously and centrifuge 5 min at 16,200 x g.
    Collect the upper phase, add 800 μl of absolute ethanol and 40 μl of sodium acetate 3 M (pH 5.0). Invert the tubes twice and incubate at -20 °C for 1 h to allow DNA precipitation.
  4. Centrifuge at least 30 min at 16,200 x g in a cold centrifuge (4 °C). Discard the supernatant and wash with 500 μl of 70% ethanol. Discard the supernatant and dry the pellets for 5 min in the speed-vac.
  5. Open a new bottle of pure water. Resuspend thoroughly the pellets in 20 μl of pure water and store DNA extracts at -20 °C for up to a few weeks, or at -80 °C for up to a few months.
  6. Digest DNA extracts by the DpnI Fast Digest restriction enzyme to eliminate possible bacterial plasmid contamination. Add 4 μl of Fast Digest 10x buffer and 1 μl of the enzyme to the 5 μl of DNA extracts. Incubate at 37 °C for 15 min.
  7. Incubate samples for 10 min at 95 °C to heat inactivate the enzyme, and to resolubilize DNA completely. Vortex each tube vigorously for at least 1 min.
  8. Dilute a fraction of the sample in 40 volumes of pure water in order to prevent the Fast Digest Buffer from inhibiting the subsequent PCR reaction.
  9. Under a DNA-free hood, prepare the reaction mix as follows (quantities per sample) : 1 μl of each primer, 10 μl of SYBR Green and 3 μl of pure water. Distribute the reaction mix in the wells of the 96-well qPCR plate and avoid air bubbles. Outside the hood, add the 5 μl of the template DNA to quantify. As a negative control, add 5 μl of pure water instead of DNA. For an absolute quantification, use standards of known concentration (ranging from 103 to 107 copies, for instance). Each sample and each standard is quantified in duplicate. To quantify the 《early》 HIV products, use the following primers : GGC TAA CTA GGG AAC CCA CTG and GCT AGA GAT TTT CCA CAC TGA CTA A. to quantify the 《late》 RT products, use : GGC TAA CTA GGG AAC CCA CTG and CCT GCG TCG AGA GAG CTC CTC TGG. To quantify the amounts of cells, quantify the quantities of GAPDH with the following primers : GGG AAA CTG TGG CGT GAT and GGA GGA GTG GGT GTC GTT.
  10. Cover the plate with an adhesive plastic film, centrifuge it briefly and heat-seal it.
  11. 《Early》 RT products (amplicon length: 183 bp) were quantified using the the following program: 35 cycles 10 sec 95 °C, 10 sec 57 °C, 15 sec 72 °C. For 《late》 RT products (amplicon length: 200 bp) and GAPDH (amplicon lenght : 303 bp), the PCR program is : 35 cycles, 10 sec 95 °C, 15 sec 57 °C, 15 sec 72 °C).
  12. To convert the concentration (in μg/μl) of standard plasmids to the number of copies, use the following formula :

    To calculate the amount of HIV-1 DNA per cell, divide the absolute DNA of HIV-1 DNA copies per half the amount of GAPDH copies (because each cell contain two GAPDH copies).
    Number of copies = Concentration/((size of the plasmid in bp*660)/6.02 x 1017)
  13.  A representative experiment of quantification of "early" and "late" RT products can be found in Figure 4D of Roesch et al. (2012).

Notes

  1. All pipetting steps are performed with filter tips.
  2. Prepare aliquots of pure water and store them at -20 °C. Use a new one for each experiment.
  3. The 《early》 RT products are amplified in the 5’ LTR region. The 《late》 RT products are amplified between the 5’ LTR region and the gag gene. These regions are highly conserved, thus the indicated primers allow to quantify HIV-1 DNA of many different strains, such as NL4-3. To know if a particular strain will be amplified by these primers, one should align the sequence of the strain and of the primers and check that it is identical.
  4. Unless stated otherwise, all steps are performed at room temperature.
  5. It is not absolutely required to measure the DNA concentration of the different samples, since the GAPDH normalization takes into account differences in DNA extraction efficiency.

Acknowledgments

This protocol is adapted from Roesch et al. (2012).

References

  1. Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012). Hyperthermia stimulates HIV-1 replication. PLoS Pathog 8(7): e1002792.

材料和试剂

  1. 感染HIV-1的感兴趣细胞
  2. 蛋白酶K(20mg/ml)(> 30U/mg)(Eurobio,目录号:GEXPRK01-B5)
  3. AL缓冲液(QIAEN,目录号:19075)
  4. 苯酚 - 氯仿 - 异戊醇(Sigma-Aldrich,目录号:77617-100ml)
  5. 氯仿(Sigma-Aldrich)
  6. 纯水(Life Technologies,Gibco )
  7. 绝对乙醇(Sigma-Aldrich)
  8. 3 M乙酸钠(pH 5.0)
  9. DpnI Fast Digest限制酶和反应缓冲液(Fermentas,目录号:FD1704)
  10. Platinium SYBR Green qPCR Supermix UDG(Life Technologies,Invitrogen TM ,目录号:11733-046)
  11. qPCR引物
  12. HIV-1(pNL4-3)和GAPDH(pGAPDH)的标准质粒

设备

  1. 离心机
  2. 无DNA罩
  3. Speed-vac
  4. 热封机
  5. qPCR机
  6. 96孔qPCR板(Eppendorf)
  7. 用于qPCR板的塑料膜(Eppendorf)
  8. 过滤提示

程序

  1. 收集在1.5ml Eppendorf管中感染HIV-1感兴趣的至少5×10 5个感兴趣的细胞,并在200×g离心3分钟。 弃去上清液,并在1ml无菌PBS中洗涤沉淀一次。 在200×g离心3分钟,然后将干燥的颗粒在-80℃下储存几个月。
  2. 打开一瓶新的无菌PBS。在180μlPBS中彻底重悬沉淀。加入20μl(12 U)的蛋白酶K和200μl的AL缓冲液。剧烈涡旋并在56℃下孵育1小时
  3. 加入400μl苯酚 - 氯仿。剧烈涡旋并在16,200×g离心5分钟。收集上层相并加入400μl氯仿。剧烈涡旋并在16,200×g离心5分钟。
    收集上层相,加入800μl无水乙醇和40μl乙酸钠3 M(pH 5.0)。将管颠倒两次,在-20°C孵育1小时,以允许DNA沉淀
  4. 在冷离心机(4℃)中以16,200× g离心至少30分钟。弃去上清液,用500μl70%乙醇洗涤。弃去上清液,以速度真空干燥沉淀5分钟
  5. 打开一瓶新鲜的纯净水。在20μl纯水中彻底悬浮沉淀,将DNA提取液在-20°C下储存几周,或在-80°C下储存几个月。
  6. 通过DpnI快速消化限制性酶消化DNA提取物以消除可能的细菌质粒污染。添加4微升的快速消化10x缓冲液和1微升的酶到5微升的DNA提取物。在37℃孵育15分钟。
  7. 在95°C孵育样品10分钟,以加热灭活酶,并完全再溶解DNA。剧烈涡旋每个管至少1分钟
  8. 将一部分样品稀释在40倍体积的纯水中,以防止快速消化缓冲液抑制随后的PCR反应。
  9. 在无DNA罩下,按如下(每个样品的量)制备反应混合物:1μl每种引物,10μlSYBR Green和3μl纯水。将反应混合物分配在96孔qPCR板的孔中,避免气泡。在罩外,加入5μl的模板DNA进行定量。作为阴性对照,加入5μl纯水代替DNA。对于绝对定量,使用已知浓度的标准(例如,范围从10×10至10×10 7个拷贝)。每个样品和每个标准品一式两份定量。为了定量"早期"HIV产物,使用以下引物:GGC TAA CTA GGG AAC CCA CTG和GCT AGA GAT TTT CCA CAC TGA CTA A.为了定量"晚期"RT产物,使用:GGC TAA CTA GGG AAC CCA CTG和CCT GCG TCG AGA GAG CTC CTC TGG。为了定量细胞的量,用下列引物定量GAPDH的量:GGG AAA CTG TGG CGT GAT和GGA GGA GTG GGT GTC GTT。
  10. 用粘性塑料膜覆盖板,短暂离心并热封
  11. 使用以下程序定量"早期"RT产物(扩增子长度:183bp):35个循环10秒95℃,10秒57℃,15秒72℃。对于"晚期"RT产物(扩增子长度:200bp)和GAPDH (扩增子长度:303bp),PCR程序为:35个循环,10秒95℃,15秒57℃,15秒72℃)。
  12. 为了将标准质粒的浓度(μg/μl)转换为拷贝数,使用下式:

    为计算每个细胞的HIV-1 DNA的量,除绝对值 HIV-1 DNA的DNA拷贝每一半量的GAPDH拷贝(因为 每个单元格包含两个GAPDH副本)。
    拷贝数=浓度/((以bp计的质粒大小×660)/6.02×1017 )
  13.  "早期"和"晚期"RT产物的定量化的代表性实验可以在Roesch等人(2012)的图4D中找到。

笔记

  1. 所有移液步骤都使用过滤嘴。
  2. 准备等分的纯水,并将其存储在-20°C。 对每个实验使用一个新的。
  3. "早期"RT产物在5'LTR区域中扩增。 "晚期"RT产物在5'LTR区和gag 基因之间扩增。 这些区域是高度保守的,因此所指示的引物允许定量许多不同菌株例如NL4-3的HIV-1 DNA。 要知道特定菌株是否会被这些引物扩增,应该对齐菌株和引物的序列,并检查它是否相同。
  4. 除非另有说明,所有步骤在室温下进行
  5. 测量不同样品的DNA浓度不是绝对必需的,因为GAPDH标准化考虑了DNA提取效率的差异。

致谢

该协议改编自Roesch等人(2012)。

参考文献

  1. Roesch,F.,Meziane,O.,Kula,A.,Nisole,S.,Porrot,F.,Anderson,I.,Mammano,F.,Fassati,A.,Marcello,A.,Benkirane, Schwartz,O。(2012)。 热疗刺激HIV-1复制 。 PLoS Pathog 8(7):e1002792。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Roesch, F. and Schwartz, O. (2013). Quantification of HIV-1 DNA. Bio-protocol 3(7): e417. DOI: 10.21769/BioProtoc.417; Full Text
  2. Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012). Hyperthermia stimulates HIV-1 replication. PLoS Pathog 8(7): e1002792.




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