Lipid-DNA Method

This page describes an alternative to the 'gelatin-DNA' method in which the lipid-based transfection reagent ('lipid') is pre-mixed with the DNA before printing. In the 'gelatin-DNA' method the lipid-based transfection reagent is added to all the samples once they have been printed on the slide. The 'lipid-DNA' method allows greater flexibility in optimizing conditions for different cell lines. We find that Effectene and Lipofectamine 2000 work reasonably well with this protocol.

Gelatin Preparation

Materials

  1. Dissolve gelatin powder in sterile MilliQ water by gently swirling mixture for 15 minutes in a 60ºC water bath. Cool the 0.2% gelatin solution at room temperature, and, while still warm (~37-40ºC), filter it through a 0.45 µm cellular acetate membrane (CA). We usually prepare 100 ml at a time and store 50 ml aliquots of the filtered gelatin solution at 4ºC.
  2. Dilute 0.2% to 0.05% using MIliQ water and store at 4ºC.

Notes on DNA Purification

DNA can be purified via any method that gives supercoiled plasmid DNA with a 260/280 absorbance ratio greater than 1.7. To purify many plasmids in parallel, we use the Qiagen Turbo Miniprep Kit. We seed bacterial clones in 1.3 ml of terrific broth (TB) in a 96-Deep-Well Plate and shake them at 250 rpm for 18-24 hours at 37ºC. Of course, DNA of equivalent or better quality can be purified via other methods and is also suitable for making transfected cell microarrays.

Sample Preparation, Microarray Printing, and Cell Addition

Materials

 

Sample Preparation

  1. In a 1.5 ml tube, add 0.80-1.60 µg DNA to 15 µl of DNA-condensation buffer (Buffer EC from Effectene Kit) in which sucrose has been dissolved to a concentration of 0.2-0.4M. The sucrose in the 'lipid-DNA' mix is necessary to stabilize the Effectene (or other lipid-based transfection reagent) after printing.
  2. Add 1.5 µl of Enhancer solution (from Effectene Kit), mix the contents by pipetting up and down five times, and incubate the mixture at room temperature for 5 minutes.
  3. Next add 5 µl Effectene transfection reagent (from Effectene Kit), mix the solution with gentle vortexing.
  4. Incubate at room temperature for 10 minutes.
  5. Add a 1X volume of 0.05% gelatin, remix the solution and pipet 20 µl into a well of a 384-well plate. 

 

Notes on Microarray Printing and Slide Storage

After transferring to a 384-well plate, we print samples onto GAPS slides with a PixSys5500 Robotic Arrayer equipped with Telechem's ArrayIt Stealth Pins (SMP4). The pins transfer the 'lipid-DNA' solution to the slide while touching the surface of the slide for 50-500 ms (this method requires longer times than the 'gelatin-DNA' method). We print spots 300-400 µm apart from other spots. To prevent clogging of the pins we maintain a 55% relative humidity environment. After printing, slides are stored at room temperature in a vacuum desiccator with anhydrous calcium sulfate pellets. Cells can be added to printed slides as soon as one hour after printing.

Cell Addition

Cells are prepared and added exactly as in the 'gelatin-DNA' method.

 

Shown below is an example of a transfected cell microarray made from a grid of lipid-DNA spots in 0.2M sucrose prepared as described above. This example shows a comparable level of reverse transfection for spots arrayed on both the GAPS-coated (Corning, Inc.) and the poly-lysine coated (Polysciences, Inc.) slides. The panels on the right are blow-ups of the boxed spots on the corresponding left panels. We calculated the DNA concentrations in the figure by dividing the total DNA amount by the final sample volume.

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*Table of Contents *Sabatini Lab *Whitehead Institute for Biomedical Research*