" . Methods Useful in Genetics and Oncology
Materials
Equipment LightCycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany)
LightCycler Capillaries (Roche Molecular Biochemicals)
ABI Prism Genetic Analyzer 310 (PE Biosystem, ForsterCity, USA)
Kits FastStart DNA Master Hybridization Probes Kit (Roche Molecular Biochemicals)
RNA 6000 LabChip Kit (Agilent Technologies, Boblingen, Germany)
TOPO TA Cloning, (Invitrogen, Groningen, The Netherlands)
Qiaprep Spin Miniprep Kit (QIAGEN, Hilden, Germany)
RiboProbe In Vitro Transcription System (Promega, Mannheim, Germany)
1st Strand eDNA Synthesis Kit for RT-PCR (AMV), (Roche Molecular Biochemicals)
Reagents Amplification Primers (Metabion, Munich, Germany)
Hybridization Probes (TIB MOLBIOL, Berlin, Germany)
DNase I (Roche Diagnostics, Mannheim, Germany)
Trizol reagent (GIBCO BRL, Eggenstein, Germany)
BamHI restriction enzyme (Roche Molecular Biochemicals)
Procedure
Sample Preparation Expression analysis of ABCAI transcripts on the LightCycler instrument was performed using different cell types grown by standard culture methods. The following in vitro cultured cells were used: THP-l cells (obtained from ATCC), HeLa
cells, keratinocytes (NHEK-Normal Human Epidermal Keratinocytes), HaCaT
cells (spontaneously immortalized human keratinocyte cell line) and fibroblasts.
Monocytes were obtained from healthy normolipidemic volunteers by leukapheresis and were purified by counterflow elutriation and were further differentiated into macrophages in the presence of human recombinant M-CSF. The basal
expression of ABCAI mRNA, the effect of differentiation (adipocyte, monocyte,
keratinocyte, HaCaT), effects oflipid loading and deloading (macrophage, THP-l
cells, fibroblasts) and its regulation by transcription factors (experiments with
Spl- and Sp3-transfected HeLa cells) were studied. Total RNA from all cultured
cells was isolated with Trizol reagent (Sigma). All RNA samples were treated with
DNase I (Roche Diagnostics) according to the protocol of Huang et al. [12). Total
RNA from different human tissues was obtained from Clontech (Heidelberg, Germany). Human subcutaneous preadipocytes and adipocytes were kindly provided by Prof. Georg Loffler (Institute for Biochemistry, Genetics and Microbiology,
University of Regensburg, Germany).
The concentration, purity and integrity of total RNA from all samples was
assessed using the Agilent 2100 bioanalyzer and the RNA 6000 LabChip Kit (Agilent Technologies). Briefly, the procedure is based on labelling of the RNA with a
fluorescent dye and, upon electrophoretic separation in capillaries embedded in
the chip, the 18S and 28S RNA peaks are identified. The presence of very distinct
and sharp peaks for 18S and 28S and a ratio of -2 (28S/18S) is indicative for high
Materials
Equipment LightCycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany)
LightCycler Capillaries (Roche Molecular Biochemicals)
ABI Prism Genetic Analyzer 310 (PE Biosystem, ForsterCity, USA)
Kits FastStart DNA Master Hybridization Probes Kit (Roche Molecular Biochemicals)
RNA 6000 LabChip Kit (Agilent Technologies, Boblingen, Germany)
TOPO TA Cloning, (Invitrogen, Groningen, The Netherlands)
Qiaprep Spin Miniprep Kit (QIAGEN, Hilden, Germany)
RiboProbe In Vitro Transcription System (Promega, Mannheim, Germany)
1st Strand eDNA Synthesis Kit for RT-PCR (AMV), (Roche Molecular Biochemicals)
Reagents Amplification Primers (Metabion, Munich, Germany)
Hybridization Probes (TIB MOLBIOL, Berlin, Germany)
DNase I (Roche Diagnostics, Mannheim, Germany)
Trizol reagent (GIBCO BRL, Eggenstein, Germany)
BamHI restriction enzyme (Roche Molecular Biochemicals)
Procedure
Sample Preparation Expression analysis of ABCAI transcripts on the LightCycler instrument was performed using different cell types grown by standard culture methods. The following in vitro cultured cells were used: THP-l cells (obtained from ATCC), HeLa
cells, keratinocytes (NHEK-Normal Human Epidermal Keratinocytes), HaCaT
cells (spontaneously immortalized human keratinocyte cell line) and fibroblasts.
Monocytes were obtained from healthy normolipidemic volunteers by leukapheresis and were purified by counterflow elutriation and were further differentiated into macrophages in the presence of human recombinant M-CSF. The basal
expression of ABCAI mRNA, the effect of differentiation (adipocyte, monocyte,
keratinocyte, HaCaT), effects oflipid loading and deloading (macrophage, THP-l
cells, fibroblasts) and its regulation by transcription factors (experiments with
Spl- and Sp3-transfected HeLa cells) were studied. Total RNA from all cultured
cells was isolated with Trizol reagent (Sigma). All RNA samples were treated with
DNase I (Roche Diagnostics) according to the protocol of Huang et al. [12). Total
RNA from different human tissues was obtained from Clontech (Heidelberg, Germany). Human subcutaneous preadipocytes and adipocytes were kindly provided by Prof. Georg Loffler (Institute for Biochemistry, Genetics and Microbiology,
University of Regensburg, Germany).
The concentration, purity and integrity of total RNA from all samples was
assessed using the Agilent 2100 bioanalyzer and the RNA 6000 LabChip Kit (Agilent Technologies). Briefly, the procedure is based on labelling of the RNA with a
fluorescent dye and, upon electrophoretic separation in capillaries embedded in
the chip, the 18S and 28S RNA peaks are identified. The presence of very distinct
and sharp peaks for 18S and 28S and a ratio of -2 (28S/18S) is indicative for high