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Molecular
Biology Protocols |
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4. RNA Analysis with the Agilent 2100
Bioanalyzer
“New Protocol – April 2003”
Caution
Note:
Cleanliness and care in setting up your experiment are absolutely
necessary for useable results. Calibrate
your micropipets daily by weighing water.
Think as you do all of your pipetting. Do not allow yourself to be distracted during
this process. Observe every
pipetting process carefully to make sure you have obtained and dispensed the
desired amount. Wear fresh gloves as you
handle the microcentrifuge tubes and set up the reactions. Think clean!
Use fresh pipet tips for each addition to avoid contaminating your RNA,
primers, and reagents.
1. Dilute total RNA samples: If the RNA concentration is expected to be
higher than 0.5 μg/μl, dilute 10-fold. Be sure that the RNA solution is well mixed
and completely dissolved. Using the L2
micropipette, add exactly 2 μl of the RNA solution to 18 μl of
nuclease-free water in a 0.5-ml microcentrifuge tube. Mix well by vortexing and spin briefly.
2. Get a tube of Ambion RNA Ladder from the
–80C freezer (box marked Towle 2002).
Each 0.5-ml tube contains enough for two analyses. Also remove the RNA chip reagents from the
refrigerator to equilibrate at room temperature in the dark for 30 min before
use. Keep the reagents in the dark as
much as possible, since the dye is light sensitive. Turn on the Agilent 2100 Bioanalyzer and
start the Agilent 2100 Bio Sizing software, selecting the Eukaryote Total RNA
Nano assay.
3. Heat both the diluted samples and the ladder
at 70C for 2 minutes (program 70-2 in the thermal cycler).
4. Clean the Bioanalyzer electrodes by adding
350 μl Ambion RNAse-Zap to a cleaning chip. Carefully insert the chip in the correct
orientation into the instrument and gently close the lid. After 1 minute, replace the cleaning chip
with one containing 350 μl nuclease-free water. Close the lid for about 10 seconds, then open
and remove the chip. Wait at least 10
seconds for the electrodes to dry. Mark
the two chips for re-use. After five
runs or at the end of the day, whichever comes first, empty the cleaning chips
into the sink and replace with fresh RNAse-Zap or water before re-use. (Note that each box of RNA chips contains
just two cleaning chips.)
5. Filter the gel matrix solution by placing 550
μl of the gel matrix (red cap) into a spin filter. Centrifuge at 4000 rpm for 10 minutes in the
Eppendorf microcentrifuge. Discard the
filter. Aliquot 65 μl filtered gel
into 0.5-ml microcentrifuge tubes included in the kit. Store the aliquots at 4C and use them within
one month of preparation.
6. Vortex the dye concentrate (blue cap) for 10
sec before use and centrifuge briefly.
Add 1 μl of dye to a 65-μl aliquot of filtered gel. Vortex thoroughly and store in the dark. Spin gel-dye for 10 minutes at 14,000 rpm in
a microcentrifuge. Use prepared gel-dye
mix within one day.
7. Make sure that the adjustable clip on the
syringe control of the Chip Priming Station is set to the upper position and the base plate is at position C. Replace the syringe when opening a new
Reagent Kit. Place a new RNA Nano chip
into the Chip Priming Station.
G
8. Add 9 μl of the gel-dye supernatant
(avoiding any pellet) to the well marked placing the pipette tip at
the bottom of the well and slowly dispensing the gel-dye without adding
bubbles.
9. Set the syringe plunger at 1 ml, then close
the Chip Priming Station cover, making sure it is securely latched.
10. Press the plunger until it is held in place
by the syringe clip. Wait for exactly 30
seconds, then release the plunger with the clip release trigger.
11. Wait for 5 seconds, then pull back the
plunger to the 1-ml position and open the cover. Turn the chip over to check for bubbles in
the microfluidic lines. If bubbles
exist, repeat step 10.
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12. Pipette 9 μl of the gel-dye supernatant
into each of the two wells marked
placing the pipette tip as described in 8.
13. Dispense 5 μl of the RNA 6000 Nano
Marker solution (green cap) into the well marked with the ladder symbol and
into each of the 12 sample wells, placing the pipette tip as described in
8. Use the chip within 5 minutes to
prevent problems due to evaporation.
14. Load 1 μl of the RNA ladder solution
into the well marked with the ladder, using the L2 micropipette, placing the
pipette tip at the bottom of the well.
Do not use the ‘blowout’ mode of the pipette.
15. In the same way, load 1 μl of diluted
sample into each of the 12 sample wells.
Do not leave any wells empty. Add
1 μl of the RNA 6000 Nano Marker (green) to the 5 μl in any unused
sample wells.
16. Firmly insert the chip into the adapter of
the IKA vortexer and vortex for 1 minute at the 2400 set point.
17. Place the chip in the Agilent 2100
bioanalyzer, making sure that the chip is inserted properly. The chip fits only one way. Do not use force. Carefully close the lid and start the run by
clicking on “Start”. During
the analysis, do not disturb the bioanalyzer or jar the lab bench on which it
sits.
18. Enter the name of the file, and click
Start. You can then complete the sample
name table while the assay is running.
If the error message Poor Chip Performance occurs, there is not
enough liquid in the wells. Prepare
another chip and make sure that the chip priming has worked and the full amount
of liquid is dispensed from each pipette tip into the wells. Do not leave any wells empty.
19. After about 30 minutes, you can check the
results of the assay. The RNA ladder
should show one marker peak at the far left and six clear RNA ladder
peaks. Samples should show the marker at
the far left, two large ribosomal RNA peaks (or three for crustacean samples),
and a heterogeneous set of minor peaks representing mRNAs. There should be little material in the early
part of the scan (degraded RNA), nor should there be substantial material in
the latest part of the scan (genomic DNA).
The software will report the concentration of RNA based on comparison
with the ladder.
Courtesy of Agilent
(http://www.chem.agilent.com)
20. After each assay, remove the chip and
discard. Clean the electrodes with RNAse-Zap and nuclease-free water as in Step
4.
21. Once a week, carefully remove and clean the
electrodes with a soft toothbrush and RNAse-free water. Dry the electrodes thoroughly before reusing.