Bio-Rad Experion RNA Analysis Kits - Manual (Page 52)

48 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Appendix A: How the Experion ™ System Works
The Experion automated electrophoresis system performs electrophoresis of samples within a
microfluidic chip. Within each chip, a series of microchannels connects the sample wells to a separation
channel and buffer wells. A set of electrodes in the electrophoresis station applies a voltage across
the microchannels, causing charged molecules in the samples to migrate into and through the
separation channel. Samples are run sequentially, with a sufficient lag between them to prevent cross-
contamination. For separation, the microchannels are filled with a proprietary gel-stain solution (GS) that
acts as a sieving matrix; therefore, the sample RNA fragments migrate through the separation channel
at a rate based on their size and charge
1 . Finally, the fragments interact with a fluorescent dye during
separation and are detected as they pass a laser and photodiode detector (laser-induced fluorescence).
RNA analysis is accomplished with the Experion RNA StdSens and HighSens analysis kits, which
perform analyses based on the following sample types:
n
mRNA
n
Total RNA — eukaryotic
n
Total RNA — prokaryotic
Overall, RNA analysis with the Experion system involves the following steps:
n
Preparing the chip (priming and loading) — Priming fills the microchannels of the
microfluidic chip with GS, which contains both the sieving matrix and fluorescent dye.
RNA samples and loading buffer are then added
n
Running the chip — The chip is inserted into the electrophoresis station, and as the
instrument lid is closed, electrodes come into contact with the solution in the wells. Voltage
is applied to the sample wells of the chip, causing the charged RNA fragments to migrate
into the separation channel. In the separation channel, the different fragments separate
as they move at different rates through the gel matrix, depending on their size. During
separation, the fluorescent dye intercalates between the bases of the fragments
n
Detecting the fragments — As the fragments migrate toward the end of the separation
channel, a laser excites the dye, causing it to fluoresce if it is bound to RNA. A photodiode
detects the fluorescence, and Experion software plots the fluorescence intensity vs. time to
produce an electropherogram and a virtual gel image
n
Analyzing the data — Following separation, Experion software subtracts background noise,
removes spikes, identifies and integrates peaks, and assigns their sizes, concentrations,
and quality (RNA quality indicator, RQI, value for eukaryotic total RNA). Following analysis,
parameters may be changed and the data reanalyzed
How Experion Software Analyzes RNA Fragments
Data Presentation
As the photodiode detects fluorescent signals from the dye-RNA complexes, Experion software
converts the signal into an electropherogram (a plot of fluorescence vs. time). Experion
electropherograms generally have the following features (Figure A.1):
n
Sample peaks — signals generated by the sample fragments
n
Lower marker — signal generated by the lower marker, which is included in the RNA
loading buffer to normalize the separation of fragments across all wells in a chip
1 RNA samples are heat-denatured to minimize the effects of shape or structure on migration.
Experion Automated Electrophoresis System