Labs which frequently perform microarray analysis usually have an Agilent Bioanalyzer to assess the integrity of RNA preparations. Agilent’s system involves the Bioanalyzer itself, a computer, the software, a priming station, an IKA vortex mixer, the kits and some other reagents. Protein or DNA chips can also be used with the system. In terms of RNA, different kits are available depending upon the amount of RNA available. I have been working with both RNA 6000 Nano LabChip Kit which uses as little as 5ng of total RNA and also with RNA 6000 Pico LabChip Kit which can work with 200pg of total RNA. I was very happy when the latter was introduced because it is the only kit which gives accurate results with extremely low RNA concentrations, like those provided after laser capture microdissection (LCM). The qualitative range for the total RNA assay is 200-5000pg/ul. The results are shown in gel-like image, electropherogram and tabular formats. The system automatically calculates the ratio of ribosomal RNA to total RNA for each sample. All results are stored and can be reviewed at any time. The most important advantage of this system is the small amount of sample used, leaving the rest of the RNA for other applications.
The RNA 6000 Pico Kit includes 25 RNA Pico Chips, 3 electrode cleaners (it is critical to clean before and after using the equipment), dye concentrate, pico marker, conditioning solution and gel matrix, 4 spin filters and a syringe. The only reagent that has to be purchased from elsewhere is the RNA 6000 ladder (from Ambion). The ladder is stored at -80C, but its quality decreases over time even when stored as recommended. If that happens, it’s a good idea to buy a new vial even though the price is high. The ladder needs to be heated at 70C for 2 minutes and diluted using RNase-free water (dilution is not necessary for the 6000 Nano Kit). The preparative steps for the gel and the gel-dye mix are very important and need to be done according to the protocol provided. The dye needs to equilibrate to room temperature (~30 minutes) and has to be covered as it is sensitive to light. Loading the gel-dye mix using the Chip Priming Station may be challenging for a beginner, but it is easy to learn. I recommend practicing by loading a few expired chips. As a hint, when the loading is done correctly, the grid on the bottom of the chip can no longer be seen. (If one looks at the bottom of the chip before loading, the grid can be easily seen.) It is critical to avoid bubbles in the chip wells. To avoid bubbles when pipetting, place the tip of the pipette near the bottom of the well when dispensing. The loading of the other reagents (conditioning solution, pico marker and ladder) and samples have to be done carefully and in a timely manner. The chip needs to be vortexed using an IKA vortexer. The vortexing step is sometimes tricky as the chip may move from the vortex. I usually attach the chip to the vortex using 2 pieces of tape, one on the top and one on the bottom. That works well. The chip needs to be run within 5 minutes of preparation. If bubbles are inside the grid, an error will show on the screen and the chip will not run on the machine.
When an RNase-free work environment is maintained and all steps of the protocol are followed exactly as suggested by the manual, then the results should be accurate and will provide the desired details about the integrity of the RNA. Always run a positive control (a sample that is known to give good results with this method) with new samples. In this way one can make sure that everything is working well.
Gertrude-Emilia Costin, PhD
Senior Research Scientist
Avon Products, Inc.
R&D, New Technology Department
Suffern, NY