Bioanalyzer

preparation of material

• 12 samples per chip
• quantitative range 25–500 ng/μl
• quantitation accuracy 20%CV (for ladder as sample)

cleaning, gel preparation (start 40min before experiement)

• take out filtered gel aliquot and fluorescent dye from fridge next door 30min ahead of time (1 gel aliquot tube enough for 2 chips)
• take out ladder from fridge upstairs
• vortex dye 10s and spin down
• mix one tube gel aliquot with 1µl of dye
• vortex, centrifuge 15000g +-20% for 10min
• wash electrodes 1min RNase ZAP, 3min water (pipette into 1 well, will spread)

prepare chip for measurement

• take out new RNA chip (pico or nano) from drawer below microarray machine and put into station
• fill 9µl gel into dark circle G well (gel reservoir)
• press down plunger and wait 30sec (gel moves through channels)
• after 30sec release silver trigger (should jump up well above 0.3, meaning seal was tight)
• wait 5sec, pull plunger up all the way
• open priming station, add 9µl gel to light circle G wells
• 5µl of marker into all sample wells and ladder well, bottom right (contains small marker to align plots)
• 1µl of sample per well, add 1µl of water or replicates into free wells (6µl required per well for machine to run properly)
• 1µl of ladder into ladder well
• vortex in holder for 1min and put into machine

start the run

• start 2100 expert software
• choose programme for kit: DNA/RNA pico/RNA nano and material: total RNA/mRNA, prokaryotic/eukaryotic
• start run

wash, export data

• typical RNA nano run takes just over 20min
• wash with clean water for 3min immediately after the run has ended (electrodes in the lid will quickly deteriorate otherwise)
• to export data as PDF you have to print; (PDF option checked will save file, PDF unchecked will print on paper)

A few typical problems occur once in a while:

• small RNA marker is not properly (often the next peak is selected => fragments size mislabelled)

cleak peak table, a tab at the bottom of the window; lower marker can be manually set here

• 18S, 28S not properly assigned (=> RIN number incorrect)