39 Biotechnology & Immunology


The ultrapure water was produced using arium® pro VF (Figure 2) as described in Nitzki and Herbig [3].


Cell Culture


PC12-tet-OFF (PC12-OFF) cells (Clontech) were cultivated in DMEM medium (glucose concentration of 1 g/L, Lonza) on poly-L-lysine-coated plates at 37°C in an incubator


with an air atmosphere humidifi ed by ultrapure water and containing 5% CO2. The medium was supplemented with 10% Fetal Bovine Serum (FBS), 5% Horse Serum (HS), 1% penicillin streptomycin and 1% GlutaMAX (Invitrogen).


Transfection of Assay Reporter Vectors


PC12-OFF cells were transfected in suspension with assay reporter vectors. For this purpose, the cells were trypsinized, pelleted and resuspended in DMEM (supplemented with only 1% FBS) with a density of 106 cells per mL. DNA-Lipofectamine complexes were prepared with 1 µg of DNA and 4 µL of Lipofectamine 2000 (Invitrogen) in aliquots of 100 µL OptiMEM Medium per 1.5 x 106 cells. After 20-minute incubation at room temperature, the mixture was combined with the cell suspension and incubated at 37°C for 4 hours. To remove the transfection reagents, the cells were washed once with DMEM medium (1% FCS). Before analysis, the cells were cultivated under identical conditions. For the assays, 5 ng per plasmid DNA and per EXT exporter construct were used. Three to fi ve reporter constructs were cloned with different EXTs for each cis-regulatory element.


Figure 2: Current arium® pro VF ultrapure water system (source: Sartorius Lab Instruments) Purifi cation of Sequencing Probes


RNA Extraction RNA was purifi ed using a Qiagen RNeasy kit along with on-column DNase-I digestion according to the manufacturer’s protocol. After RNA purifi cation, the probes were precipitated by one-half volume 7.5 M ammonium acetate (prepared using ultrapure water) and three-fold volume of 100% ethanol. For cDNA synthesis, 1 µg of extracted RNA, Superscript III Reverse Transcriptase (Invitrogen) and a 120 pmol random nanomer primer were used. The samples were tested for their RNA purity, and possible DNA contamination was excluded by running controls that did not contain any reverse transcriptase (-RT).


Amplifi cation of EXT-cDNA Products by PCR for Decoding


Decoding PCR runs were performed using a HotStarTaq Plus DNA polymerase, dNTPs (both supplied by Qiagen) and ultrapure water. Thirty 3-step cycles (30 sec. at 95°C, 30 sec. at 59°C and 30 sec. at 72°C) were run (Figure 3). Reverse-transcribed cDNA served as the starting material. No cDNA was used in the negative control (last lane).


Coding PCR runs were carried out also using HotStarTaq Plus DNA polymerase and ultrapure water; however, only ten 3-step cycles were run (cycle steps: 30 sec. at 95°C; 20 sec. at 58°C; 20 sec. at 72°C) (Figure 3). For each Coding PCR, 1 µL of a 1:10 diluted Decoding PCR sample was utilised as the starting material; no cDNA was used in the negative control. All PCR products were visualised and tested by agarose gel electrophoresis.


Sequencing According to the Semiconductor Method


EXT reporter molecules were sequenced by NGS using an Ion PGMTM machine (Figure 4). The sequencing technology of this machine is based on sequential detection of protons that are released during the polymerisation process of DNA. Depending on the base to be polymerised (adenine, guanine, cytosine or thymine) on the complementary strand, the corresponding dNTP is incorporated and a proton is released, which is detected by ion sensors [4].


Sequence Analysis


Individual UNIX-based tools from the FastX Toolkit [5] were used to analyse the sequencing data (FASTQ-to-FASTA conversion program and FASTX barcode splitter). The presence and identity of each EXT was determined by BLAST (Basic Local Alignment Search Tool) analysis [6] against a self-prepared reference library of EXTs, and differentially expressed EXTs were identifi ed and quantifi ed using R scripts [7].


Figure 5: Workfl ow for purifi cation of sequencing samples and the use of ultrapure water for the individual steps


Article fi rst published in Lab Asia 24.1 January/February


Figure 4: Ion Torrent Personal Genome Machine® (Ion PGMTM sequencer) (source: laboratory at Systasy Bioscience GmbH, Munich, Germany).


Results


The multi-parametric assay platform was used in combination with NGS and ultrapure water to measure a large number of downstream cell signalling responses that occur after a) a specifi c stimulus, b) a broad stimulus or c) after the addition of a specifi c inhibitor. In this case, PC12 cells were transfected with 33 different EXT reporters, which represent the activities of eleven cellular signalling pathways. To apply a specifi c stimulus (a), the ERBB4 receptor was transfected. This receptor plays a role in many different biological processes, including heart development and differentiation of neurons, as well as in associated human diseases, such as cancer and psychiatric disorders, including schizophrenia [8,9]. The ERBB4 receptor can be activated by its ligand Neuregulin 1, or by the biologically active and soluble domain of Neuregulin 1, EGF-like domain (EGFld), and multi-parametric assays were previously used to monitor the activities of the ERBB4 receptor [1,10]. Further, ERBB4 signalling activity mediated by EGFld was specifi cally blocked by the ERBB kinase inhibitor lapatinib (c), which is also used as a cancer therapy drug. A combinatorial stimulus consisting of phorbol-12-myristate-13-acetate (PMA) and serum was selected as a broad stimulus (b).


Figure 3: Quality control of EXT-amplifying PCR steps. Agarose gel runs of A) Decoding PCR and B) Coding PCR. The last lane of each run indicates the negative control with ultrapure water; primer dimers are visible in the last lanes.


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