Bio-Rad Experion Protein Analysis Kits - Manual (Page 27)

Appendix A — How the Experion ™ System Works
The Experion 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
fragments migrate through the separation channel at a rate based on their size and charge.
Finally, sample fragments interact with a fluorescent dye during separation and are detected
as they pass by a laser and photodiode detector.
RNA analysis is accomplished with the Experion RNA analysis kits. Two RNA analysis kits
are available, and they differ in the level of sensitivity that they offer: the Experion RNA
StdSens analysis kit is used for the evaluation and quantitation of samples containing as
little as 5 ng/µl RNA, and the Experion HighSens analysis kit is used for higher sensitivity
evaluation of as little as 100 pg/µl RNA. Both kits use the following steps for RNA analysis:
1. Preparing the chip (priming and loading) –– Priming fills the microchannels of the
microfluidic chip with the GS, which contains both the sieving matrix and fluorescent
dye. RNA samples are then prepared and added to the sample wells along with the
loading buffer.
2. Vortexing the chip –– Vortexing ensures that samples and loading buffer are mixed.
Vortexing the chip itself reduces the number of sample manipulation and pipetting
steps required, improving reproducibility.
3. Running the chip –– The chip is inserted into the electrophoresis station and, as the
instrument lid is closed, the electrodes come in 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 RNA fragments
separate as they move at different rates through the gel matrix, depending on their
size. During separation, the fluorescent dye intercalates into the RNA strands.
4. Detecting the fragments –– As the fragments migrate towards the end of the separation
channel, a laser
4 excites the dye, causing it to fluoresce if it is bound to RNA (excess,
free dye does not fluoresce appreciably). A photodiode detects the fluorescence, and
Experion software plots the fluorescence intensity versus time to produce an
electropherogram and a virtual gel image.
5. Analyzing the data –– Following separation, Experion software subtracts background
noise, removes spikes, identifies and integrates peaks, and assigns their sizes and
concentrations. Following analysis, parameters may be changed and the data reanalyzed.
The software also assigns 18S and 28S rRNA peaks based on their size and calculates
the total concentration of the sample by comparing the area under the sample
electropherogram (excluding the lower marker) to the area under the ladder
electropherogram.
4 635 nm excitation, 685 nm filter collection.
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