Bio-Rad Experion RNA Analysis Kits - Manual (Page 55)

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Data Analysis: Normalization, Sizing, Quantitation, and Quality Determination (RQI)
Following separation, Experion software analyzes RNA fragments using one or more of the following:
n
Internal 50 bp lower marker for normalizing the migration times of samples in different wells
n
RNA ladder for determining fragment size (sizing) and concentration (quantitation)
n
RNA quality indicator (RQI, eukaryotic total RNA analysis only) to quantitate the state of
degradation of a sample
Normalization: Aligning Peak Migration Times
To compensate for small variations in each separation, Experion RNA analysis uses an internal 50 bp
marker to normalize the migration times among samples. The marker is included in the Experion RNA
loading buffer and so is added to each sample and the RNA ladder (Figures A.1–A.3). Inclusion of the
marker and the normalization process ensures that the system software properly identifies peaks.
RNA Sizing: Determining Peak Sizes
The first sample to be analyzed is the RNA ladder, which contains 8 RNA fragments of 200–6,000 nt.
Experion software constructs a standard curve of migration time as a function of fragment 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: Determining RNA Amount
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 the corrected area, the known ladder concentration
(160 ng/μl for the Experion RNA StdSens assay and 1,000 pg/μl for the Experion RNA HighSens assay),
and a correction factor to determine the RNA concentration of the sample. Results are displayed under
the Results and Run Summary tabs (Figures A.4 and A.5).
Quality Determination (RQI)
The RQI algorithm compares the electropherogram of RNA samples to data from a series of
standardized, degraded RNA samples and automatically returns a number between 10 (intact RNA)
and 1 (degraded RNA) for each sample (Figures A.4 and A.5 and Table 5.1). RQI analysis is performed
for samples with an Experion StdSens or HighSens analysis kit. The RQI feature of Experion software
allows you to quantitate the level of degradation in a eukaryotic total RNA sample. Refer to bulletin 5761
for more details about RQI.
Fig . A .5 . Run Summary tab . RQI results appear with quantitation results under the Results (Figure A.4) and
Results Summary tabs. Example shown is for a total RNA Experion RNA HighSens analysis.
Experion RNA StdSens and HighSens Analysis Kits