The RetroSys™ RT Activity Kit
Product code: RT-001

Protocol for Quantification of RT activity.

20 cell supernatant samples in four dilutions may be analysed with one kit.

1. Collect kit components.
• Take out the following components from the refrigerator:

  2 PolyA Plates
  1 Sample Dilution Buffer (bottle B)
  2 RT Reaction Components (vial C1)
  1 Reconstitution Buffer (bottle C2)
  1 HIV-1 rRT-standard (vial D)
  1 Concentrated Washing Buffer (bottle E)
  4 pieces of adhesive tape
  

2. Prepare the reaction mixture.
• Add 12 ml of Reconstitution Buffer (C2) to each of the two vials containing RT Reaction Components (C1). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Transfer the contents of both C1 vials to a 50 ml glass beaker.
• Add 12 ml of distilled water and mix thoroughly.

  
  

3. Prepare the PolyA Plates.
• Take out both PolyA Plates from their foil pouches. Save one foil pouch for later use.
• Add 150 µl of reaction mixture (beaker) to each well of both plates.
• Seal the plates with adhesive tape.
• Incubate the plates at 33°C for 20-60 minutes.

  
  

4. Dilute the HIV-1 rRT standard.
   When preparing the standard dilutions, mix thoroughly and change pipette tips between each transfer.
• Add 6 ml of Sample Dilution Buffer (B) to the vial with HIV-1 rRT-standard (D). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Add 250 µl of Sample Dilution Buffer (B) to twelve test tubes.
• Add 100 µl of HIV-1 rRT-standard (D) to the first of the twelve test tubes.
• Transfer 200 µl from the first test tube to the second.
• Transfer 200 µl from the second test tube to the third and so on until a series of twelve dilutions has been obtained.

  
  

5. Dilute the samples.
   Avoid aspirating cells when sampling cell culture samples.
• Add 160 µl of Sample Dilution Buffer (B) to all wells in columns 1-10 of a sample preparation plate (96-well mictrotiter plate, not supplied).
• Add 40 µl of the first sample to be analysed to well A1.
• Add 40 µl of the second sample to be analysed to well A2 and so on until all 20 samples have been added to wells A1-10 and E1-10.
  
  Dilution of samples in well A1-10.
  Mix thoroughly and change pipette tips between each transfer.
• Transfer 40 µl from wells A1-10 down to their corresponding B-wells.
• Transfer 40 µl from wells B1-10 down to their corresponding C-wells.
• Transfer 40 µl from wells C1-10 down to their corresponding D-wells.
  
  Dilution of samples in well E1-10.
  Mix thoroughly and change pipette tips between each transfer.
• Transfer 40 µl from wells E1-10 down to their corresponding F-wells.
• Transfer 40 µl from wells F1-10 down to their corresponding G-wells.
• Transfer 40 µl from wells G1-10 down to their corresponding H-wells.

  
  

6. Start the RT reaction.
• Take out the PolyA Plates from the incubator.
• Transfer 50 µl of each sample dilution from the sample preparation plate to each corresponding well in columns 1-10 of both PolyA Plates.
• Add 50 µl of Sample Dilution Buffer (B) to wells E12-H12 of both plates.
• Add 50 µl of the first standard dilution to well A11 of both plates.
• Add 50 µl of the second standard dilution to well B11 and so on until all twelve standard dilutions are set on both plates.
• Seal the PolyA Plates with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the plates at 33°C on an orbital shaker set at gentle agitation.
  The first plate is incubated for 3 hours and the second overnight.

  
  

7. Prepare the wash fluid.
• Add 75 ml of Triton X-100 to 1 litre of distilled water and mix thoroughly with a magnetic spin bar.
  Make sure that the Triton X-100 dissolves completely which takes approximately 10 minutes.
• Add 10 ml of Concentrated Washing Buffer (E) to a 10 litre container.
• Add the dissolved Triton X-100 to the 10 litre container.
• Adjust the volume to 10 litres with distilled water and mix thoroughly.
  The residual Concentrated Washing Buffer (E) is used on day two and should be stored in the refrigerator until then.

  
  

8. Stop the RT reaction of the first plate.
   After 3 hours of incubation the RT reaction of the first plate is stopped by washing. Use approximately 6 ml of wash fluid per well and wash.
• Wash the PolyA Plate twice. Turn the plate 180° between washes if possible.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.
• Return the plate to its foil pouch and store it at -20°C.

  
  

  
  

Day two

9. Collect kit components.
• Take out the kit box from the refrigerator and take out the following components:

  1 Concentrated Washing Buffer (bottle E)
  2 RT Product Tracer (vials O)
  1 AP Substrate Tablets (tube P1)
  1 AP Substrate Buffer (bottle P2)
  2 pieces of adhesive tape
  2 plastic lids
  

10. Prepare the wash fluid.
    Follow the procedure for this as described in step 7.

    
    

11. Prepare the RT Product Tracer.
• Add 12 ml of distilled water to each of the two vials containing RT Product Tracer (O). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Transfer the contents of both O vials to a 50 ml glass beaker.
  

  
  

12. Stop the RT reaction of the second plate.
    After overnight incubation the RT reaction of the second plate is stopped by washing. Use approximately 6 ml of wash fluid per well and wash.
• Wash the PolyA Plate twice. Turn the plate 180° between washes if possible.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.
• Collect the first PolyA Plate from the freezer.
  

  
  

13. Start the binding of RT Product Tracer.
    When dispensing the tracer, pipette tips should not touch the bottom of the wells.
• Add 100 µl to each well of both PolyA Plates.
• Seal the plates with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the plates for 90 minutes at 33°C on an orbital shaker set at gentle agitation.

  
  

14. Prepare the AP substrate solution.
• Add the AP Substrate Tablets (P1) to the AP Substrate Buffer (P2).
• Shake the bottle occasionally and the tablets will dissolve completely in approximately twenty minutes.

  
  

15. Remove excess tracer.
    After 90 minutes of incubation, excess tracer is removed by washing. Use approximately 6 ml of wash fluid per well and wash.
• Wash both PolyA Plates four times. Turn the plates 180° between washes if possible.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.

  
  

16. Start alkaline phosphatase reaction.
    The AP substrate solution must be mixed thoroughly and equilibrated to room temperature before it is added to the PolyA Plates. When dispensing the substrate solution, pipette tips should not touch the bottom of the wells.
• Add 200 µl of AP substrate (P2) solution to each well of both PolyA Plates.
• Cover the plates with plastic lids and incubate them at room temperature under dark cover on an orbital shaker set at gentle agitation.

  
  

17. Read the plates.
    Set the filter of the plate reader to 405 nm.
• Read the absorbance of both plates after 30 minutes of incubation.
• Read the absorbance of both plates a second time after 2 hours of incubation.
• Read the absorbance of both plates a last time after overnight incubation.
  

  
  

18. Process the data.
    The RetroSys™ RT assay gives a linear relationship between A₄₀₅ and RT activity if the readings are within the linear measuring range of the plate reader used
    When calculating the results the data from each reading time should be processed separately, i.e. values for standard curve and samples should be from the same reading time.
• Plot the A₄₀₅ of each standard dilution (wells A11-D12) against its concentration of HIV-1 rRT (see table below). Preferentially use a computer program to calculate the best regression line utilising the mean A₄₀₅ of the background controls (wells E12-H12) as y-axis intercept. Only use readings within the linear measuring range of the microtiter plate reader.
• Determine the RT activity of each well with the aid of the regression line. Only determine RT activity for wells giving an A₄₀₅ within the linear measuring range of the reader and greater than two times the mean of the background controls.
• Calculate the RT activity for undiluted sample by compensating for the dilution used in the assay.
• Calculate the mean RT activity of the sample. Should significantly lower values be obtained for higher sample concentrations, this is due to disturbing factors in the sample. Values obtained from these dilutions should be excluded from the calculation.