Bio-Rad Experion RNA Analysis Kits - Manual (Page 47)

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Error Probable Cause Recommended Action
No peaks detected in
some samples There is not enough sample in the wells Ensure pipets are calibrated and that correct volumes have
been added to the wells
Samples were not prepared properly Review the sample preparation procedure detailed in
Chapter 3
RNA stain was photobleached Protect RNA stain and gel-stain solution (GS) from light
Prepare new GS; use a new tube of RNA stain if necessary
Particulates are clogging the microchannels Use only high-quality 0.2 µm-filtered water (such as
Experion DEPC-treated water, not autoclaved water)
Verify that gel (G) and GS were properly filtered
If using samples that contain particulates, perform a quick
microcentrifuge spin of the prepared sample to pellet
particulates before loading
Remove solutions from the top of the liquid to prevent
introducing particulates pelleted during centrifugation
No peaks detected in any
samples Gel-stain solution (GS) is old or photobleached Prepare fresh GS
Chip was primed with incorrect pressure and
time settings Check settings on the priming station and repeat analysis
with a new chip
Particulates are clogging microchannels Use only high-quality, 0.2 µm-filtered water (such as
Experion DEPC-treated water, not autoclaved water)
Verify that gel (G) and GS were filtered properly
If using samples that contain particulates, perform a quick
microcentrifuge spin of the prepared sample to pellet
particulates before loading
Remove solutions from top of liquid to prevent introducing
particulates pelleted during centrifugation
Lower marker is missing Samples do not contain loading buffer or were
not prepared properly Review the sample preparation procedure in Chapter 3
Electropherogram peaks are
much smaller than expected
(peaks are <50% of what is
expected) Not enough sample was added Ensure pipets are calibrated to accurately deliver ≤10 µl
Too much salt in sample; salt ions compete
with sample ions during injection Ensure RNA is in buffer of low ionic strength (for RNA
StdSens use TE or water; for RNA HighSens use water)
Analyze a dilution series of sample in water on another
chip. If peak heights and concentrations of diluted sample
increase, salt is too high in the original sample. Determine
proper dilution to minimize salt effect
Gel-stain solution (GS) is old or photobleached Prepare fresh GS
Reagents, specifically RNA stain, were not
brought to room temperature prior to use; if
stain is not equilibrated to room temperature, it
will be more concentrated when it is added to
gel, which may result in lower peaks and areas Repeat analysis with a new chip, new tube of stain, and
properly equilibrated reagents
Filtered gel (G) or gel-stain solution (GS) were
mislabeled or used improperly. RNA chips
contain wells for GS that carry sample through
microchannels and supply dye for analysis Ensure chip was primed with GS
Ensure G was applied to well labeled G and GS was used in
all wells marked GS
Experion RNA StdSens and HighSens Analysis Kits