外周血单核细胞中DNA总量及 2-LTR （长末端重复序列） HIV DNA、HIV RNA 和 疱疹病毒 DNA 的量化

Marta Massanella; Sara Gianella; Steven M. Lada; Douglas D. Richman; Matthew C. Strain

doi:10.21769/BioProtoc.1492

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Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs

外周血单核细胞中DNA总量及 2-LTR （长末端重复序列） HIV DNA、HIV RNA 和疱疹病毒 DNA 的量化

Marta Massanella email

Sara Gianella

SL Steven M. Lada

DR Douglas D. Richman

MS Matthew C. Strain email

发布: 2015年06月05日第5卷第11期 DOI: 10.21769/BioProtoc.1492 浏览次数: 13973

评审: Vaibhav B ShahVamseedhar RayaproluAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Journal of Virology, featuring study using the protocol.

Feb 2014

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Abstract

Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood mononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR).

For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to protocol “Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma” (Vargas-Meneses et al., 2015).

Keywords: HIV reservoir (HIV病毒库)

Droplet Digital PCR (ddPCR) (液滴数字PCR（mRNA）)

CMV (CMV)

EBV (EB病毒)

Materials and Reagents

Cell processing and flow cytometry
1. 1x Dulbecco’s phosphate buffered saline (DPBS) (Corning, catalog number: 21-031-CV )
2. RPMI 1640 medium (Life Technologies, Gibco^®, catalog number: 11875-093 )
3. GemCell U.S. origin fetal bovine serum (FBS) (Gemini Bio Products, catalog number: 100-500 )
4. 37% formaldehyde solution (Sigma-Aldrich, catalog number: F8775-500ML )
5. Sodium azide Reagents Plus (NaN3) (Sigma-Aldrich, catalog number: S2002-25G )
6. Antibodies
  1. CD3-APC (BD, catalog number: 340440 )
  2. CD4-FITC (BD, catalog number: 347413 )
  3. CD45-PerCP-Cy5.5 (BD, catalog number: 340953 )
7. AllPrep DNA/RNA Mini Kit (QIAGEN, catalog number: 80204 )
8. Staining flow cytometry buffer (staining buffer) (see Recipes)
9. 1% formaldehyde (1% FA) buffer (see Recipes)
10. RLT plus buffer + β-ME (see Recipes) (included in the AllPrep DNA/RNA Mini Kit)
11. 70% ethanol buffer (see Recipes)
12. 10 mM Tris elution buffer (see Recipes)
DNA/RNA co-extraction and DNA precipitation
1. RNase-free DNase set (QIAGEN, catalog number: 79254 )
2. AllPrep DNA/RNA Mini Kit (QIAGEN, catalog number: 80204)
3. Molecular biology grade sterile purified water (RNase, DNase, proteinase free)
4. Ethyl alcohol pure (200 Proof molecular biology grade) (Sigma-Aldrich, catalog number: E7023 )
5. 3 M NaOAc (sodium acetate) (Affymetrix, catalog number: 75897 )
6. UltraPure glycogen (Life Technologies, catalog number: 10814010 ) (concentration: 20 µg/µl)
7. 2-Mercaptoethanol (β-ME) (Life Technologies, Gibco^®, catalog number: 21985-023 )
8. Tris hydrochloride (1 M solution pH 8.0 molecular biology grade) (Thermo Fisher Scientific, catalog number: BP1758-500 )
9. RNase-free DNase set (see Recipes)
ddPCR
1. Ban-II restriction enzyme (New England Biolabs, catalog number: R0119L )
2. Primers and probes (Table 1, IDT.) (Final PCR concentration: 900 nM primer, 250 nM probe)
3. iScript advanced cDNA synthesis kit (Bio-Rad Laboratories, catalog number: 172-5038 )
4. ddPCR supermix for probes (no dUTP) (Bio-Rad Laboratories, catalog number: 1863024 )
5. Droplet generator cartridges and gaskets (Bio-Rad Laboratories, catalog number: 1864007 )
6. Droplet generator oil (Bio-Rad Laboratories, catalog number: 1863005 )
7. Droplet reader oil (Bio-Rad Laboratories, catalog number: 1863004 )
8. 2x buffer control (Bio-Rad Laboratories, catalog number: 1863052 )
9. Pierceable foil heat seal (Bio-Rad Laboratories, catalog number: 1814040 )
10. Multichannel reagent trough
11. Molecular biology grade sterile purified water (RNase, DNase, proteinase free)

Equipment

Cell processing and flow cytometry
1. 96-well plate V-bottom (VWR International, catalog number: 12-565-460 )
2. Water bath at 37 °C
3. BD Accuri (with plate loader) (BD, catalog number: 653118 ) or equivalent flow cytometer
4. Sorvall^TM RC4 general purpose floor model centrifuge (Thermo Fisher Scientific, catalog number: 75004481 ) or equivalent
DNA/RNA co-extraction and DNA precipitation
1. Collection tubes (2 ml) (QIAGEN, catalog number: 19201 )
2. BD 3 ml Leur-Lok^TM Syringe with detachable BD 23G 1½ PrecisionGlide^TM needle (BD, catalog number: 309589 )
3. BD 20G 1½ PrecisionGlide^TM needle (BD, catalog number: 305176 )
4. Sterile 1.5 ml microcentrifuge Eppendorf tubes
5. Sterile 1.5 ml Micro tubes with screw cap
6. Pasteur pipette (transfer pipette)
7. Vortex
8. Heating block
9. Table centrifuge
10. NanoDrop 2000 Spectrophotometer
ddPCR
1. 96-Well PCR clean plate
2. Strip caps
3. Plate sealing adhesive film
4. 96-Well semi-skirted PCR plate (Eppendorf, catalog number: 951020362 )
5. Low retention pipette tips (200 µl and 20 µl)
6. 1.5 ml screw-top tubes
7. Biorad droplet generator and reader, QX200 droplet digital PCR system (Bio-Rad Laboratories, catalog number: 186-4001 )
8. PX1 PCR plate sealer (Bio-Rad Laboratories, catalog number: 181-4000 )
9. Thermocycler
10. Special multichannel pipette for ddPCR droplet transfer (P-50) (e.g. Rainin, catalog number: L8-50XLS+ )
11. Centrifuge to spin plates
12. Spectrophotometry (Nanodrop)

Procedure

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Massanella, M., Gianella, S., Lada, S. M., Richman, D. D. and Strain, M. C. (2015). Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs. Bio-protocol 5(11): e1492. DOI: 10.21769/BioProtoc.1492.