12 samples per chip
quantitative range 25–500 ng/μl
quantitation accuracy 20%CV (for ladder as sample)
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)
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 2100 expert software
choose programme for kit: DNA/RNA pico/RNA nano and material: total RNA/mRNA, prokaryotic/eukaryotic
start run
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)