Bio-Rad Experion RNA Analysis Kits - Manual (Page 20)

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2. Evaluate the separation of the RNA ladder. To display the ladder electropherogram, click the ladder
well in the project tree, or click on the lane labeled L in the virtual gel. The electropherogram should
resemble the one shown in Figure 3.6 and should have the following features (if your ladder does not
have these features, see Chapter 7, Troubleshooting for more information):
n
Lower marker peak at ~20 sec, at least 5 fluorescence units above the baseline, and
distinguishable from the ladder peaks
n
Eight resolved ladder peaks
n
Flat baseline
Fig . 3 .6 . Experion RNA StdSens separation of the RNA ladder in a prokaryotic total RNA assay . Note the flat baseline and resolved peaks.
RNA ladder peaks
Baseline
Lower marker
3. Examine the separation of at least one sample. Click on the sample name in the project tree or on
the lane in the virtual gel to view the electropherogram, which should have the following features:
n
Lower marker peak at ~20 sec, at least 5 fluorescence units above the baseline, and
distinguishable from the sample peaks
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Sample peaks: mRNA analyses often yield broad peaks and, on occasion, contaminating
18S and 28S rRNA peaks (Figure 3.7), while total RNA analyses often yield 2–3 prominent
peaks corresponding to rRNA (in eukaryotic sources, 18S and 28S and occasionally 5S; in
prokaryotic sources, 16S and 23S) (Figure 3.8)
n
Flat baseline
If the lower marker peaks are not properly assigned, manually set the marker (see Section 6.1).
4. Evaluate the data analysis performed by Experion software (see Chapter 4, Data Analysis). If
necessary, change the analysis settings and parameters by following the instructions in Chapters 5
and 6.
Experion Automated Electrophoresis System