Running RNA samples on the Agilent BioAnalyzer
For examination of 1 µl RNA samples (25-500 ng/µl Total RNA, 25-250 ng/µl mRNA).
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 the sample wells and load the ladder
- 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.
keep gel-dye mix in the dark
gel-dye mix should be used within 1 day of preparation
Filtered gel should be used within one month of preparation