Glossary of Terms 320
V4.2 LabChip GX User Manual PerkinElmer
RNA Assay Analysis
RNA analysis initially progresses similarly to DNA analysis except
that the baseline for RNA peaks is calculated using a spline-fit,
much like the baseline used for the Baseline Subtraction option.
However, this computed fit is not subtracted from the data; instead it
is used to determine the peak height and to limit the peak extends.
The RNA ladder is similar to the DNA ladder but does not contain
an upper marker. RNA sample data is aligned with the lower maker
and then the sample peaks are sized using point-to-point
interpolation between ladder peaks. The most prominent peaks
found within predefined size windows are identified as ribosomal
rRNA genes 5S, 18S and 28S (for eukaryote RNA), 16S and 23S
(for prokaryote RNA).
For the purpose of quantitizing the full RNA content in the sample, a
straight-line baseline is drawn across the bottom of the sample by
finding the local averages about its endpoint. The area baseline
endpoints can be moved on the Graph View after the “Show Peak
Baselines” option is selected in the Graph View Properties.
Trapezoidal integration from the straight-line baseline to the data
signal is used to calculate the total RNA area. Integration is
performed from the end of the lower marker to the endpoint of the
area baseline. The range from the end of the 5S to the start of the
18S peak is termed the Fast Area. This area is calculated in the
same way as the total RNA area.
For the purpose of quantitizing the rRNA peaks, a two-point
baseline is drawn across the bottom of each peak identified as
rRNA and the area is computed. These areas and rRNA peak
heights and some relevant ratios are available for display in the
Well Table View. A combination of the quantities is used to assess
the quality of the RNA sample.
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