Quality assurance of RNA derived from,,,laser microdissected tissue samples,,,obtained by the PALM MicroBeam System,,,using the RNA 6000 Pico LabChip kit.
Comparison of staining
In this test serial tissue
sections were stained using
(HE), Nuclear Fast Red (NFR),
Methylgreen (MG) or Methylene
Blue (MB). Areas corresponding
to 2000 cells were dissected,
the RNA purified using Manufacturer
B (3 replicates each)
and analyzed with the 2100
bioanalyzer (figure 5A) and
the Light-Cycler as above.
The cDNA input for the PCR
corresponded to a fourth of
the RNA amount used for the
2100 bioanalyzer run. The melting
curve proves the specificity
of the products (figure 5B),
while the growth curve reflects
the starting amount of RNA
(figure 5C). In contrast to the
test with Manufacturer C (see
above) NFR was the optimal
stain, though the Cp-values lay
very close together (only half
a cycle difference) except
for MB which showed worse
results. (CpNFR: 34,7; CpHE:
35,2; CpMG: 35,8; CpMB: 37,6).
This real-time RT-PCR results
again correlated well with the
2100 bioanalyzer values.
RNA derived from laser-microdissected
tissue isolated by the
PALMMicroBeam system is of
high quality and can be analyzed
efficiently using the RNA 6000
Pico assay with the Agilent 2100
bioanalyzer. RNA-purification kits
of different manufacturers and
various common staining procedures
have been tested and were
compatible with the Pico Assay.
Nevertheless, it seems to be advisable
that any extraction protocol
and staining method should be
tested in combination to find
the optimal procedure for tissue
The RNA 6000 Pico kit is well
suited to show differences in RNA
quality and, therefore, is an ideal
tool to optimize experimental
conditions. Its unprecedented
sensitivity allows for the first
time quality control in the context
of microdissection experiments
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