Bio-Rad Experion Protein Analysis Kits - Manual (Page 31)

Data Analysis: Normalization, Sizing, and Quantitation
Following separation, RNA fragments are analyzed using one or more of the following:
• An internal marker for normalizing the migration times of samples in different wells
• RNA ladder for determining fragment size (sizing) and concentration (quantitation) of
samples
Normalization: Aligning the Peaks
To compensate for small variations in samples or factors influencing separation (pH, dye
concentration, porosity of matrix, injection volumes, etc.), Experion RNA analysis uses an
internal marker to normalize the migration times between samples. The 50 bp lower marker
is included in the RNA loading buffer and, therefore, appears in all samples. Inclusion of this
marker and the normalization process ensures that the system software properly identifies
peaks. Once aligned, the software then further analyzes the data as described below.
Sizing
The first sample to be analyzed is the RNA ladder, which has been optimized for automated
electrophoresis on the Experion system. The RNA ladder contains eight purified 200–6,000 nt
RNA fragments. Experion software constructs a standard curve of migration time as a
function of size from the RNA ladder separation. It then calculates the size of the RNA
fragments from the sample wells by comparing their migration times to the standard curve.
Quantitation
Experion software begins RNA quantitation by comparing the time-corrected area (corrected
area) under the electropherogram of a sample (excluding the lower marker) with the corrected
area under the electropherogram of the RNA ladder. It then uses this value and the known
ladder concentration (160 ng/µl for the RNA StdSens assay, and 1,000 pg/µl for the RNA
HighSens assay) to determine the RNA concentration of the sample.
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