Running RNA samples on the Agilent BioAnalyzer

Remove gel-dye mix from from fridge, or prepare and date a fresh batch. Equilibrate mix to Room Temp before use.

clean the electrodes

use 350 µl RNAseZAP in special rinse chip (can be re-used within the day)

incubate 1 minute

Rinse with 350 µl H2O for 10 seconds. Open the lid and wait 10 seconds before putting anything else in.

Prepare Chip

• Place new chip on the Chip Priming Station
• Place 9 µl Gel-dye mix in well marked G (with black circle)
• Place syringe plunger at 1 ml
• Close Priming Station and push plunger down until clip is engaged
• Wait 30 seconds and release plunger.
• Check the underside of the chip for air bubbles. If bubles are seen, repeat pressurization.
• Place 9 µl Gel-dye mix in the other two wells marked G

• Place 5 µl RNA 6000 Nano Marker (use 6 µl for wells that will not contain any sample).
• Place 1 µl RNA 6000 ladder into the lower right well.
• Load 1 µl of each Sample into the appropriate well

Run the chip

• Place the chip in the Vortexer and shake for 1 minute.
• Place the chip in the BioAnalyzer and close the lid.
• Select the mRNA Nano option from the Assay Menu.
• Click the Start... icon to begin the run.

Preparation of RNA gel-mix (4 chips)
Allow gel and dye to equilibrate to Room Temp for 30 minutes

• Prepare Filtered Gel Aliquots
  • Aliquot 550 µl of gel to spin filter, and spin at 1500g (~4000 RPM) for 10 minutes.
  • Aliquot 65 µl filtered gel to new tubes.
  • Date and label the tubes. The filtered gel aliquots must be used within 1 month.

• Prepare Gel Dye Mix
  • vortex the dye
  • Combine 1 µl dye with 65 µl aliquot of gel; vortex.
  • Spin at 13000g for 10 min. (~14000 RPM)
  • Label the tube clearly (i.e. Gel/Dye Mix) and use within one day of preparation. Avoid pipeting particulates near the bottom of the tube.