Genomic DNA was isolated from eukaryotic cell lines using the QIAamp DNA Mini Kit (Qiagen, The Netherlands). Cells were detached from cell culture plates by adding 0.05%
trypsin. The reaction was quenched with 5 ml medium and cells were centrifuged at 300 x g for 4 min. The cell pellet was washed with 3 ml phosphate-buffered saline (PBS) and centrifuged again, the supernatant was discarded. The cell pellet was resuspended in 200 µl PBS and transferred to a 1.5 ml microcentrifuge tube. A volume of 20 µL Proteinase K and 200 µL AL buffer were added to the cell pellet, mixed vigorously for 15 sec and incubated at 56°C for 10 min. Subsequently, 200 µl ethanol p.a. was added and lysate was mixed vigorously. The lysate was loaded onto the spin columns placed in a 2 ml collection tube. The samples were centrifuged at 6,000 x g at room temperature for 1 min. The collection tube was replaced with a fresh microcentrifuge tube. A volume of 500 µl AW1 buffer was added to wash the sample and samples were centrifuged at 6,000 x g at room temperature for 1 min. The collection tube was replaced with a fresh microcentrifuge tube. 500 µl of AW2 buffer was added to wash the sample. The samples were centrifuged at 20,000 x g at room temperature for 3 min. The
collection tube was replaced with a fresh microcentrifuge tube and centrifuged again to remove residual buffer for 1 min. The spin column was placed into a fresh microcentrifuge tube. To elute the DNA, 2x50 µl of RNase-free water was added to the middle of the column, incubated for 1 min and subsequently centrifuged for 1 min to collect DNA. The DNA concentration was quantified using the NanoDrop™ spectrophotometer (Thermo Fisher Scientific, USA) as described in section 3.4.3.1.
3.4.2 RNA extraction
Total RNA was isolated from cell lines using the RNeasy Mini Kit (Qiagen, The Netherlands) and RNA concentrations were measured using the NanoDrop™ system (Thermo Fisher Scientific, USA) as described in section 3.4.3.1. RNA from adherent cells was harvested with 600 µl RLT buffer supplemented with 6 µl β-mercaptoethanol, scraped from cell culture plates and stored at -80°C until isolation. If tumor tissue was used, powdered tissue was lysed in 600 µl RLT buffer supplemented with 6 µl β-mercaptoethanol and pipetted up and down until roughly dissolved. For both sample types, one volume of 70% ethanol was added to each sample, mixed by pipetting and 700 µl of lysate was loaded onto the RNeasy spin columns placed in a 2 ml collection tube. The samples were centrifuged at 15,871 x g at room temperature for 1 min.
Flow-through was discarded. This procedure was repeated until all sample was loaded onto the column. A volume of 700 µl RW1 buffer was added to wash the column, followed by centrifugation and removal of flow-through. Subsequently, 500 µl of RPE buffer were added to the columns and they were centrifuged for 1 min. The flow-through was discarded. Another 500 µl of RPE buffer was added to the columns followed by subsequent centrifugation. To remove residual buffer, columns were put into a fresh collection tube and centrifuged for 1 min.
The collection tube was replaced with a fresh microcentrifuge tube. To elute the DNA, 2x15 µl of RNase-free water was added to the middle of the column, incubated for 1 min and subsequently centrifuged for 1 min to collect RNA. The RNA concentration was quantified using the NanoDrop™ spectrophotometer (Thermo Fisher Scientific, USA) as described in section 3.4.3.1.
Methods 54
3.4.3 Nucleic acid quantification
3.4.3.1 Measurement of RNA or DNA concentration with NanoDrop™
To determine the RNA or DNA concentration, the absorbance spectrum was measured with the NanoDrop™ 2000c spectrophotometer (BioTek). The device was blanked with RNase-free water and 1 µl of purified RNA or DNA was taken for analysis and was loaded to the sample plate. The NanoDrop™ software calculates nucleic acid concentrations by analyzing the absorption spectra at 260 und 280 nm.
3.4.3.2 Measurement of DNA concentration with Qubit™
For sensitive measurements of DNA for the ChIP and WGS Sequencing and the methylation array, the Qubit® fluorometer (Thermo Fisher Scientific, USA) was applied. The working solution was prepared by diluting the Qubit® dsDNA HS Reagent 1:200 in Qubit® dsDNA HS Buffer. For the two supplied calibration standards, 190 µl of working solution and 10 µl of standard were mixed. For each sample, 199 µl of working solution was mixed with 1 µl of sample DNA. Samples were incubated at room temperature for 2 min. Standards were measured from 300-1000 nm wavelength and samples at 260 nm. Concentration of samples was calculated as followed:
Concentration of sample = Measured value × 200
3.4.4 Reverse transcription
The First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used to transcribe complementary DNA (cDNA) from RNA for qRT-PCR analysis. Per reaction, 0.1-1 µg of total RNA was diluted with RNase-free water to a total volume of 10 µl, subsequently 1 µl of random hexamer primer mix was added. The annealing of primers to RNA was performed at 65°C for 5 min in a PCR cycler. Next, 4 µl 5x reaction buffer, 1 µl RiboLockTM RNase Inhibitor (20 u/µl), 2 µl of 10 mM dNTP Mix and 2 µl of M-MuLV Reverse Transcriptase (20 u/µl) were added to each reaction. The cDNA synthesis was performed in accordance to described PCR program (Table 25) and complementary DNA was stored at -20°C until use.
Table 25: PCR program applied for cDNA synthesis.
Temperature Duration
Incubation 25°C 5 min
Synthesis 37°C 60 min
Denaturation 70°C 5 min
3.4.5 Quantitative real-time polymerase chain reaction
Relative gene expression was measured using SYBR Green Dye (Roche, Switzerland) on a StepOne Plus or QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, USA), and was normalized to the averaged expression of 18S, HRPT or SDHA (Fischer, 2005). All primers used in this study are summarized in Table 11. For quantitative real-time polymerase chain reaction (qRT-PCR) analysis, 18 µl of master mix was added to each 96-well. The composition of master mixes was adapted to corresponding primer type (Table 26). Complementary DNA was diluted 1:10 with RNase-free water and 2 µl of the diluted cDNA was used for analysis.
Table 26: Reaction mixture for qRT-PCR analysis.
Self-designed Primer
(100 pmol) 1x Quantitect Primer (forward & reverse Mix) 1x
PCR Mix 2x 10 µl 10 µl
RNase-free H2O 7.84 µl 5.8 µl
Primer Forward: 0.08 µl
Reverse: 0.08 µl 2.2 µl
cDNA 1:10 2 µl 2 µl
The quantitative real-time PCR was performed using the following temperature protocol (Table 27):
Table 27: PCR program applied for qRT-PCR analysis.
Temperature Duration Cycles
50°C 2 min 1
Denaturation 95°C 10 min 1
Annealing 60°C 1 min
Extension 95°C 15 sec 40
95°C 15 sec 1
Melt curve 60-95°C + 0.3°C increments
Methods 56
Data was analyzed using StepOne™ v2.3 or QuantStudio™ v1.5.1 Software (Thermo Fisher Scientific, USA). Changes in expression were calculated using the ΔΔCt method for the cell culture experiments or the standard curve method for in vivo experiments. Each primer was validated before use and tested by using the standard curve method (VanGuilder, 2008).