Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR

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, Invitrogen^TM, 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, Invitrogen^TM)
   
3. Primers sense and antisense (Biomers)
   
4. Isolated RNA
   
5. TOPO TA Cloning Kit (Life Technologies, Invitrogen^TM, 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, Invitrogen^TM, 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