The RetroSys™ C-type RT Activity Kit
Product code: RT-011

Protocol for Quantification of RT activity.

2 x 20 cell supernatant samples in four dilutions may be analysed on one kit.

1 Prepare the reaction mixture.

• Add 12 ml of Dilution Buffer (B) to one vial containing C-type RT Reaction Components (C). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Add 6 ml of distilled water and mix thoroughly.
  
  

2 Prepare the PolyA Plates.

• Take out the PolyA Plate (A) from its foil pouch.
• Add 150 µl of reaction mixture to each well of the plate.
• Seal the plate with adhesive tape.
• Incubate the plate at 33°C for 20-60 minutes on an orbital shaker set at gentle agitation.
  
  

3 Dilute the MuLV RT standard.
When preparing the standard dilutions, mix thoroughly and change pipette tips between each transfer.

• Add 4 ml of Dilution Buffer (B) to the vial with MuLV RT standard (D). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Add 250 µl of Dilution Buffer (B) to seven test tubes.
• Add 250 µl of MuLV RT standard (D) to the first of the seven test tubes.
• Transfer 250 µl from the first test tube to the second.
• Transfer 250 µl from the second test tube to the third and so on until a series of seven dilutions has been obtained.
  

 

4 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.

 

5 Start the RT reaction.

• Take out the PolyA Plate from the incubator.
• Transfer 50 µl of each sample dilution from the sample preparation plate to each corresponding well in columns 1-10 of the PolyA Plate.
• Add 50 µl of Dilution Buffer (B) to wells H11-H12.
• Add 50 µl of the first standard dilution to well A11 and A12.
• Add 50 µl of the second standard dilution to well B11 and B12 and so on until the seventh standard dilution is set in wells G11 and G12.
• Seal the PolyA Plate with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the plate at 33°C for 3 hours on an orbital shaker set at gentle agitation.
  

6 Prepare the wash fluid.

• Add 15 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 2 ml of Concentrated Washing Buffer (E) to a 2 litre container.
• Add the dissolved Triton X-100 to the 2 litre container.
• Adjust the volume to 2 litres with distilled water and mix thoroughly.
  

 

7 Prepare the RT Product Tracer.

• Add 12 ml of distilled water to one vial containing RT Product Tracer (O). Make sure that the lyophilised material dissolves properly and mix thoroughly.

 

8 Stop the RT reaction.
After 3 hours of incubation the RT reaction is stopped by washing.

• Wash the PolyA Plate according to wash protocol.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.

 

9 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 the PolyA Plate.
• Seal the plate with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the plate for 90 minutes at 33°C on an orbital shaker set at gentle agitation.

10 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.
  
  

11 Remove excess tracer.
After 90 minutes of incubation, excess tracer is removed by washing.

• Wash the PolyA Plates according to wash protocol.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.
  
  

12 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 the PolyA Plate.
• Cover the plate with a plastic lid and incubate at room temperature under dark cover on an orbital shaker set at gentle agitation.

 

13 Read the plates.
Set the filter of the plate reader to 405 nm.

• Read the absorbance of the plate after 1 hour of incubation.
• Read the absorbance of the plate a second time after 2 hours of incubation.
• If necessary, read the absorbance of the plate after prolonged incubation.

 

14 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 the mean value of each standard dilution (wells A11-A12 to G11-G12) against its concentration of MuLV RT (see table below). Preferentially use a computer software to calculate the best regression line utilising the mean A₄₀₅ of the background controls (wells H11-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.