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Normalization and Re-array

(Last updated: April 2020)

After IVT, dsRNA concentrations vary across each plate. Normalization is necessary to ensure that all wells in each plate have the same concentration of dsRNA. A re-array of the reagents in each 96-well plate into quadrant plates is also needed so that each stamped out assay-ready plate will have the desired layout. We use nuclease-free water (CAS# 7732-18-5, US Biological Life Sciences) to normalize the dsRNA and to fill in any empty wells.

Organizing robot files

1. Send Claire (DRSC bioinformatics team) the list of designs and controls, along with any user specifications (empty wells around the borders to avoid edge effects, any controls the user may want to add themselves etc.). Make sure to send the IVT list with location information on where each design is located on the IVT plates.

2. After receiving the plate layout file from Claire, send it to Stephanie (DRSC Director) and the user for final approval.

  • Check the file to make sure the quadrant plate re-array makes sense. If making an assay-ready plate with the two empty outer rings of wells to prevent edge effects, the quadrant plates should each have one empty outer ring of wells because of the stamping protocol in the Bravo. See the Operating the Bravo protocol for more details.

3. After receiving approval for the layout, create the robot files for the four quadrant plates.

  • This will involve a lot of copy/pasting from Claire’s layout file to the new robot files. Be very careful that all the information is transferred correctly.
  • See the MultiProbe and Normalization protocol for instructions on how to determine the amount of sample and water needed per well .
  • Carefully transfer the volume information onto the robot files.
  • Because the concentrations of the dsRNA may be very high, it may not be possible to directly normalize to bathing (50 ng/uL) or transfection (16 ng/uL) concentrations. In this case, normalize to a convenient concentration such as 150 ng/uL or 200 ng/uL. Then dilute the plates to the correct concentrations by hand using a new set of u-bottom quadrant plates.
  • Make sure all plates are clearly labeled and dated. These plates will all go to the user, along with PCR and IVT products.

4. Use the MultiProbe to normalize and re-array the dsRNA into quadrant plates.

  • A water reservoir is needed; see the MultiProbe and Normalization protocol on how to prepare a reservoir.
  • If the dsRNA is in a full-skirted PCR plate, change the labware on the deck view in the MultiProbe program. This will ensure the tips extend the correct distance to aspirate samples.
    • Determine where the PCR plates will be placed. Then delete the current labware at that location on the deck view by right-clicking on the plate and selecting “Remove labware from deck.”
    • Right-click in the empty space and select “Add labware…”
      1. Category: 96 well plate
      2. Labware: 96 well PCR rack (Perkin-Elmer)
      3. Support tile: Plate-Adapter support tile
      4. Name: Plate[#]
    • Make sure the name of the plate matches what is on the robot file! Otherwise, the protocol will not run.

5. After normalizing and re-arraying the reagents, seal all plates with the PlateLoc. Label and date all plates. Include the concentration on normalized plates. Store at -80.

Note: For “custom IVT” requests, we ship the PCR templates as well as the dsRNA itself. Please see related comment at the end of the IVT protocol.