Viral copy number

Determining Lentiviral copy number by qPCR

This protocol is designed for the quantification of lentiviral copies integrated in a human genome. To this end, we target both lentiviral (Psi) and human (albumin) genes with Taqman probes, and use a synthetic construct (Alb-Psi standard) for absolute

quantification (see “Oligos and Standard” for the sequences).

MATERIALS AND REAGENTS

• DNeasy Blood and Tissue Kit (Qiagen)
• Absolute qPCR MIX Low ROX Mix (Thermo Scientific)
• Oligos and standard
• Eppendorfs 1,5 ml and 0,2 ml
• Axygen^® 96-Well Polypropylene PCR Microplate
• MicroAmp Optical adhesive film (Thermo Scientific)
• Sterile water

BEFORE YOU START

      - Extract Genomic DNA using the DNeasy Blood and Tissue Kit (Qiagen).

      - Resuspend oligos at 100 µM and use these stocks to prepare 10 µM dilutions.

     - Resuspend Alb-Psi standard to a concentration of 0,005 µg/µl, then store in 10 µl aliquots.

      - In your calculations, allow for triplicates and a 10% excess.

PROCEDURE

1. Prepare 8 serial dilutions (in 10-fold increments) of Alb-Psi standard to achieve 2x10⁸ to 2x10¹ copies/µl and dilute genomic DNA down to 20ng/µl and 2ng/µl.

2. Prepare a mastermix containing (per reaction):

3. Add 5 µl of genomic DNA or Alb-Psi Standard per reaction to achieve a final volume of 12,5 µl. 

4. Seal the plate with the adhesive film and use a standard Taqman program to perform the qPCR.

50 °C, 2 min; followed by initial denaturation at 95 °C, 10 min; 40 cycles of [95 °C, 15 sec; 60 °C, 1 min]; 95 °C 10 min.

5. Analyze results in QuantStudio 3 Design and Analysis Software (Thermo Fisher Scientific). The 2^-ΔΔCt method is used to calculate the number of copies per genome normalized to albumin.

OLIGOS AND STANDARD (5'->3')

cn_Psi_F: CAGGACTCGGCTTGCTGAAG

cn_Psi_R: TCCCCCGCTTAATACTGACG

cn_hAlb_F: GCTGTCATCTCTTGTGGGCTG

cn_hAlb_R: ACTCATGGGAGCTGCTGGTTC

cn_Psi_P_FAM: [6FAM]-CGCACGGCAAGAGGCGAGG-[BHQ1]

cn_Alb_P_HEX: [VIC]CCTGTCATGCCCACACAAATCTCTCC[TAMRA]

Alb-Psi standard (only one strand is shown):

TAGAAAGTTGCTGTCATCTCTTGTGGGCTGTAATCATCGTCTAGGCTTAAGAGTAATATTGCAAAACCTGTCATGCCCACACAAATCTCTCCCTGGCATTGTTGTCTTTGCAGATGTCAGTGAAAGAGAACCAGCAGCTCCCATGAGTTTGGATAGCCtctcTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATC

Alb-Psi is 309 bp long (MW=203940 g/mol). Therefore, a 0,005 µg/µl is equivalent to 14766598019 molecules/µl.