Bio-Rad Experion RNA Analysis Kits - Manual (Page 48)

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Error Probable Cause Recommended Action
Peaks are small, broad, or
missing in some samples
but present in other samples
on the same chip Contaminants are present Prepare samples in a different buffer or dilute in water
Do not use autoclaved water for diluting samples or
ladder. Use only high-quality 0.2 µm-filtered water (such as
Experion DEPC-treated water)
RNA ladder and samples were not denatured
completely Heat samples and RNA ladder for 2 min at 70°C before
loading
Do not use RNA ladder that has been repeatedly heated and
frozen
Late migration (peaks
broaden and are delayed
over the course of the run) Turn analysis off (see Section 6.5, Turning Analysis Off, for
more information). Late migration is indicated if there is a
drift in the migration of lower marker across the chip. Refer
to the troubleshooting tips that follow
Air bubbles are interfering with electrical
contact in one or more of the wells Stop run, remove chip, and use a clean pipet tip to remove
or dislodge the bubbles, or remove and replace the solution
in affected wells
When pipetting, insert the tip vertically and to bottom of the
well. Dispense liquids slowly. Do not expel air at the end of
the pipetting step. Dispense only to the first stop
Peaks migrating faster than
usual; electropherogram
appears compressed Electrophoresis station temperature is
inappropriate or fluctuating during run (should
be 30–35°C); there is no cooling unit in the
electrophoresis station, so if the temperature
changes during the run, samples exhibit
different separation characteristics Ensure the temperature of the room is appropriate and
stable, and place the electrophoresis station away from all
heat sources, such as windows or ovens
Sizing is incorrect RNA ladder peaks and lower markers were
improperly assigned by the software Manually assign lower marker (follow instructions in Section
6.1, Manually Setting a Marker), if necessary
Quantitation is incorrect Lower marker was not properly assigned by
the software Check that lower marker was properly assigned. Manually
select the marker, if necessary (follow instructions in Section
6.1, Manually Setting a Marker)
Pipetting and/or dilution errors occurred during
sample preparation or chip loading Ensure calculations and dilutions are correct
Ensure pipets are calibrated, and use pipets that accurately
deliver volumes of ≤10 µl
Do not modify the sample preparation and loading protocols
described in this manual
Ensure the total RNA concentration of samples is within the
linear dynamic range; if total RNA concentration is unknown,
analyze a sample dilution series
Review the essential practices described in Chapter 2
before initiating another analysis with a new chip
Samples are old or have not been stored
properly and RNA degraded Prepare fresh samples and try the analysis again
Ghost peaks or
contaminants appear in
electropherograms Electrodes are contaminated Perform the deep cleaning procedure described in
Appendix B and software Help section (search term
“electrodes”)
Use only 0.2 µm-filtered water (such as Experion DEPC-
treated water) for diluting samples and the RNA ladder;
do not use autoclaved water
RNA ladder degraded Aliquot the RNA ladder for single use and store at –70°C
Experion Automated Electrophoresis System