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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

Jolien Vermeire Jolien Vermeire
Bruno Verhasselt Bruno Verhasselt

发布: 2013年06月05日第3卷第11期 DOI: 10.21769/BioProtoc.780 浏览次数: 14441

评审: Anonymous reviewer(s)

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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

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

微生物学 > 微生物遗传学 > RNA > qRT-PCR

分子生物学 > RNA > qRT-PCR

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