The Agilent Bioanalyzer offers a ‘lab on a chip’ micro-fluidics platform for analysis of DNA, RNA, protein and cell; the instrument offers sizing, quantification, and quality control, and delivers results in high quality digital data. The automated system offers a simple and easy use method to assess up to 12 samples in under an hour, from sample preparation to running the chip. Ready-to-use assays and pre-packaged reagents are available, in order to minimize hands-on time and produce readily reproducible results.
A number of kits are available for use with the Bioanalyzer. The RNA kits offer the ability to check RNA quality, including assignment of an RNA Integrity Number (RIN). Kits are available for total RNA in nano to pico amounts and also for small RNAs. I use the Bioanalyzer to assess total RNA quantity, integrity and ribosomal ratios for my samples. In addition, I use the RIN number as a standard integrity measure that is independent of concentration or instrument. I routinely use the RNA 6000 Nano Kit, which is designed to quantify total RNA from 25-500 ng/ul and will provide a qualitative value when in the 5-500 ng/ul range. The kit contains all reagents required for RNA analysis, however the user must supply RNaseZAP® (Ambion, Inc) for electrode decontamination and also RNase-free water.
With each new kit, DNA ladder and gel aliquots must be prepared, which adds time to the first use. However, once this has been done, the instrument and chip take just approximately 15 minutes to prepare and 30 minutes to run, for up to 12 samples.
In order to run a chip, the user must first prepare the gel-dye mix. This involves adding RNA 6000 Nano dye concentrate to an aliquot of filtered gel and thoroughly mixing the preparation by vortexing and centrifugation for 10 minutes. The gel-dye mix can then be added to the chip, using the supplied chip priming station. The chip fits into the station and the user should pipette 9.0µl of the gel dye mix into the appropriately marked well. It is important that the pipette tip is inserted right down to the bottom of the well at this point, in order to prevent air bubbles from forming. The user must then set the supplied timer to 30 seconds, adjust the attached syringe to 1 ml and close the chip priming station. The plunger of the syringe should be pressed down until it is held by the priming station clip and after the 30 second hold the clip should be released. After 5 seconds, the user should pull back the plunger to the 1 ml position and open the chip priming station. Following this preparation, 9.0 ul of gel-dye mix should be added into each of the marked wells. The user then pipettes 5 ul of the RNA 6000 Nano marker into the 12 sample wells, including any wells which will not be used for sample analysis. The next step is to add the samples and ladder. Only 1ul of each is required for analysis and they must all be heat denatured at 70°C for 2 minutes before use. Once the chip is fully loaded, it must be vortexed for 60 seconds in the specially designed vortex, which is included with the Bioanalyzer. The chip is then ready to run.
Following analysis, the Bioanalyzer provides both gel and elecropheretogram traces. The user can identify high quality RNA samples by one marker peak, two ribosomal peaks and a high RIN number.