204 Agilent 4150 TapeStation System Manual
10
Troubleshooting
Troubleshooting RNA Applications
Incorrect or Missing RIN
e
Value
The RNA Integrity Number equivalent (RIN
e
) is an automated numerical
software assessment which allows determination of sample integrity. It was
validated to match the RNA Integrity Number (RIN) of the Agilent 2100
Bioanalyzer system. The algorithm takes into account the peak height of the
18S peak in relation to the background signal to calculate a RIN
e
value for
total RNA.
Table 14 RNA - Possible causes for missing or incorrect RIN
e
results in order of probability.
Root cause Explanation Solution
Sample
concentration
outside
recommended range
for application
An overconcentrated sample will alter the
electropherogram significantly. A sample below
the detection limit may also not get an
assigned RIN
e
Either dilute or concentrate the sample until it
is within the recommended range for the
application as stated in the respective assay
Quick Guide (see also “RINe Functional
Range” on page 112); then prepare a new run.
Use the High Sensitivity assay if applicable
(Ta bl e 3 on page 64)
Incorrect heating
step
The 18S peak may have partially reverted back
to its original, non-denatured conformation
which results in characteristic double peaks
and influences RIN
e
calculation
Ensure the correct denaturation conditions
during RNA sample preparation, according to
the assay Quick Guide. Samples should be kept
on ice after preparation, and run within 2 hours
of denaturation (see “Heat Denaturation” on
page 99) (Table 3 on page 64)
Genomic DNA
contamination
Samples that are contaminated with genomic
DNA contain additional signals migrating in and
beyond the region of the 18S and 28S.
Occasionally this peak can be mistaken for the
18S or 28S peak
Ensure that all peaks are annotated correctly
and correct if this is appropriate. Treat samples
with DNAse to eliminate the genomic DNA
peak then prepare a new run
There are 2 bands
present at the 18S
peak position
There may have been insufficient sample
denaturation prior to analysis.
Ensure the correct denaturation conditions
during the RNA sample preparation, according
to the assay Quick Guide (Ta ble 3 on page 64).
The 18S peak may have partially reverted back
to its original, non-denatured conformation.
Samples should be kept on ice after
preparation, and run within 2 hours of
denaturation, see “Heat Denaturation” on
page 99 for more information.
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