RNA Extraction – Purification – Robotic method (96R Ext_ARN PVG100+ Purification BM)

Rev 1.0 (29/06/2017)

Reagents and Materials included in the ADNucleis kit:

  • ADNUCLEIS Viral RNA Extraction-Purification kit (Extraction kits and Purification kits are sold separately)
    • RNA extraction kit: PVG working solution must be prepared before starting the extraction step)
      • PVG (Please reconstitute the solution in this bottle)
      • PE
      • TME
      • RNA carrier (Keep at 4°C)

(Please use immediately after preparation).

  • Nucleic acid Purification kit

    • Magnetic beads suspension (µMB)
    • Precipitation buffer SP
    • Wash buffer 1
    • Wash buffer 2
    • Wash buffer 3
    • Elution buffer (ELU)
    • Decontamination buffer for the robot needles: D1, D2 et D3

Reagents and Materials not provided in the kit

  • Consumables
    • Deep-well 96 wells plate (for the robotic method,  please use the following plate: Riplate plus PP 1mL ref. n°43001-0016, Ritter brand).
    • Aluminium foil film for 96 wells plate.
    • 96 wells PCR plate
    • QPCR film for 96 wells PCR plate

The material above  is not included in the kit, but can be purchased at ADNucleis. Please contact ADNucleis to check the compatibility of your own consumables.

  • Routine laboratory reagents and materials (to be used according to the Good Laboratory Practices): tips, micropipettes (1mL, 200µL, 20 µl, 10 µl), non-powdered gloves …
  • Ultrapure water (sterile, distilled and nuclease free).
  • RNA Standard.

RNA extraction is achieved using enzymatic activities: An enzymatic and thermal lysis releases the RNA from all microorganisms and cells present in the sample. .

Preparation of the PVG working solution :

  • Resuspend PE with the appropriate volume of TME (ME) when first opening the PE vial. Homogenize
Reagents  TME volume
PE 25 mg 440 µl
PE 100 mg 1760 µl

Store at -20°C until use.

  • Prepare enough master working solution of PVG + ME (100µL of PVG for 4µL of ME) extemporaneously for X number of samples to be extracted +3 (e.g. for 20 samples, prepare a volume of master mix for the equivalent of 23 samples).
  • BE CAREFUL TO HOMOGENIZE CORRECTLY THE PVG in order to have NO volute in the end (10 times).
  • RNA carrier should be added in the mix PVG + ME at this step if extracting RNA. Add 1 µl of RNA carrier per sample (e.g. 23 µl)
Reagents 1 reaction 24 reactions 48 reactions 72 reactions 96 reactions
PVG 100 µl 2700 µl 5100 µl 7500 µl 9900 µl
ME 4 µl 108 µl 204 µl 300 µl 396 µl
RNA carrier 1µl 27 µl 51 µl 73 µl 99 µl

Volumes in the table above for 24, 48, 72, 96 reactions take into account the loss of volume due to pipetting. ADN_SOFT takes into account the loss of volume due to pipetting automatically.

  • Distribute 100µl of Extraction buffer PVG + ME (+RNA carrier if added) in each well of the deep-well plate.
  • For PIPETTOR ROBOT automated procedure, this distribution is done column by column beginning with the first column (A1, B1…H1, A2, B2…H2 etc.)

    

It is possible to have the first sample placed in any well (Offset option). However, the filling of the following samples (2,3,4,…) must follow the same rule as above: samples are distributed column by column from top to bottom and no empty well is allowed between the first and last sample. The number of empty wells before the first sample is given in the “plate Layout” sheet.

  • Homogenize samples carefully, then pipette 100µl and add to a well of the deep well plate.
  • Vortex or homogenize by pipetting the sample with the PVG.
  • Put the deepwell plate with samples on the heating block (Thermoshaker)
  • Start ADN_SOFT.exe (“START A NEW RUN”), and click immediately on “Save As..” and save the archive file in the default folder.
  • fill the plate layout with sample names (each name has to be different), then click on “Lock Layout”.
  • Specify the volume of elution buffer used for the RNA elution step.
  • The table below indicates reagent volumes to be filled in the different containers of the robot.
  • The program used for the Extraction-Purification method is “µMB Extract-Purif” :

  • Check that the volume in the Liquid System jar is sufficient (2% D3 in demineralized water). Empty the “Waste” jar.
  • Start Gemini software (Software controlling the Tecan Robot), then open the extraction purification program : ADNucleis_Extract_Purif.gem
  • Fill the different containers with reagents according to the positions showed on the Layout in Genimi software (Required volumes are given by ADN_SOFT).
  • Put an empty deepwell plate in “DW-Empty” position, This plate will be used to collect eluted RNA.
  • Start the Inheco Multitec heating block. The Gemini program controls the temperatures of the different blocks.
  • Click “flush” on Gemini software

  • Initialize the robot

  • Click “Play” in Gemini software to launch the extraction-purification program. A prompt window will ask the number of sample, fill the number of sample.

NB : the robot will start by the thermic lysis step. This steps takes about 30 min. The robot does not show any visual activity.

  • At the end of the program (30 min + 1min per sample), the RNA is eluted and the plate is kept at 4°C.
  • Go back to ADN_SOFT to start the setup of the PCR plate

 

Posted in Technical notice.