2.5. Gene expression analysisGene e

2.5. Gene expression analysis
Gene expression of several bone-specific markers (Table 1) was
analyzed by semi-quantitative real-time RT-PCR. For this purpose
total mRNA from hFOB 1.19 cells was isolated after 3, 7,
14, 21, and 28 days of culture on the different materials using
the RNeasy MiniKit (Qiagen, Hilden, Germany). Integrity and
concentration of the RNA were determined using a lab on a
chip RNA 6000 Nano Series II kit (Agilent Technologies, Waldbronn,
Germany) that was run on an Agilent 2100 Bioanalyzer
instrument (Agilent Technologies). Reverse transcription was
performed with 5 g total RNA.
The cells cultured on the discs were harvested by
trypsinization and washed with PBS. The primer sequences
of selected genes were determined with the “Universal Probe
Library” software from Roche (http://qpcr.probefinder.com/
organism.jsp). Only intron-spanning sequences were chosen
in order to exclude contamination with genomic DNA. Realtime
RT-PCR was performed in a Light Cycler 489 (Roche, Basel,
Switzerland) in a 384 plate. A 10 l volume with 5 l SYBR
Green I Mastermix (Roche), 50 ng cDNA as template and 0.5 M
of the primer-pairs were used respectively.
Light cycling conditions were as follows: activation (95 ◦C
for 10 s), 40 amplification cycles (95 ◦C for 10 s, 52 ◦C for 5 s,
and 72 ◦C for 12 s). Melting curve analysis was used to ensure
that all transcripts under investigation were represented by a
single peak, indicating specificity. Gene expression was calculated
from the real-time RT-PCR efficiency [44] in relation to
the mean of five housekeeping genes (LMNA, GAPDH, ACTB,
RPS18, and RPLP0). Three independent cultures were used for
real-time RT-PCR measurements.