Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine
© Schomaker et al.; licensee BioMed Central. 2015
Received: 10 September 2014
Accepted: 1 December 2014
Published: 3 February 2015
In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells.
The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization.
This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.
The direct modulation of gene expression is essential to establish therapeutic approaches in molecular medicine. Additionally to the development of therapies on the molecular level, the evaluation of target genes as therapeutic agents by combining the technology of RNAi and high throughput screenings is of major interest [1-3].
A major challenge in molecular medicine is the efficient, non-toxic and cell type independent transfection of cells in high throughput. In general a very effective manipulation strategy to achieve this is the transduction of cells via viral vectors. However, despite of the high efficiency this method bears high biological risk as integrational mutagenesis . Alternative existing non-viral transfection methods show specific advantages and disadvantages. Transfection with lipid based reagents is often applied in high throughput assays but this method is cell type dependent and occasionally inefficient, especially for primary- and stem cell transfection . Due to the difficulties in transfection of these cells, the commonly employed manipulation methods are either electroporation or nucleofection [6,7]. Unfortunately, these methods affect cell viability which is crucial when handling sensitive cells. Consequently in this manipulation it is essential to achieve a balance between transfection efficiency and methodical toxicity. Electroporation and nucleofection can also be utilized for high throughput assays, but these physical techniques remain usually limited to well plates with low well numbers being additionally cost ineffective .
In order to address these challenges methodicaly, a variety of optical transfection techniques have been developed based on pulsed as well as continuously emitting lasers [9-13]. None of these techniques fulfills the requirements of an efficient and low-toxic transfection method combined with high throughput. Accordingly, there is no laser based technique currently established, that allows routinely laboratory or clinical use. A promising tool for molecular medical applications is the nanoparticle mediated laser transfection using a microchip laser emitting ps laser pulses with a resonant wavelength of 532 nm [14,15]. Herein, gold nanoparticle (AuNP) labeled cells are irradiated with a weakly focused laser beam. This method allows targeting many cells simultaneously, ensuring high throughput while maintaining a high spatial selectivity. Additionally, this physical method using resonant laser pulses is very promising for the manipulation of a variety of cell types.
By applying off-resonant fs laser pulses, the transfection of hematopoietic stem cells (CD34+) can be achieved . Here, the excitation of the membrane adhered AuNP with the incident laser light leads to plasmon resonances which increase the absorption and scattering cross section of the AuNP by several orders of magnitude. When the AuNP is irradiated at a resonant wavelength, the laser energy is absorbed leading predominantly to thermal effects and changes in the particles morphology [15,17]. Using near infrared (NIR) femtosecond (fs) laser systems, off-resonant AuNP excitation can be achieved . At this wavelength the absorption and therefore the thermal impact is reduced and the incident light is scattered into the near field of the particle. Due to this “nanolens” effect, an enhancement of the electric field in the near field takes place . If the AuNP is adhered to the cell membrane, the field enhancement can initiate a spatially confined membrane permeabilization . In proof of principle experiments we could show the possibility to perforate the cell membrane using off resonant 800 nm fs laser pulses to deliver fluorescent labeled small interfering RNA (siRNA) and plasmid DNA (pDNA) into cells [20,21]. In another fs based study, a DNA-transfection rate of 23% using a melanoma cell line was stated and plasma induced nano-cavitation is supposed as the membrane permeabilization effect . The advantage of NIR wavelengths located in the “diagnostic window” regime of the electromagnetic spectrum results in higher penetration depths into biological tissue which might allow in vivo applications . Furthermore, the low absorption cross section in the NIR reduces the risk of thermal induced AuNP fragmentation.
Interaction of cells with gold nanoparticles
Scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) provided detailed information about the attachment and distribution of the AuNP at the cell membrane after co-incubation and several washing steps (Figure 2B, C). The results show a loose dispersion of AuNP after 1 h of incubation. The particles were located at the culture dish bottom and on the cell membrane. By increasing the incubation time to more than 3 hours, the particles started to aggregate at the cell membrane. After an incubation time of 5 h, no further increase could be observed. Depending on the location of the particles, some of the particles appeared brighter than others. At higher magnifications, as visible in Figure 2C, some particles were located on the cell membrane (solid ellipse Figure 2C) and some were started to be endocytosed (dashed ellipse Figure 2C), which is demonstrated by the cell membrane covered particles. Based on this we defined an incubation time of 3 h for our gold nanoparticle mediated laser transfection. Within this time a sufficient number of particles adhere to the cell membrane to induce membrane permeabilization. The number of particles at the cell membrane was counted using ESEM images of ZMTH3 cells taken after 3 h of incubation. An incubation concentration of 11 μg/ml was applied which represents the optimal concentration for cell manipulation. On average 164 ± 50 particles at the membrane of a single cell were counted.
Evaluation of efficient and gentle cell manipulation parameters
To evaluate the optimal process parameters for an efficient and gentle cell manipulation, the cells were treated with different parameters in the presence of 10 kDa FITC labeled dextran and the corresponding fluorescence level was determined. As an indicator for viability, the respective metabolic activities of the manipulated cells were measured after laser treatment using an fluorescence based assay (Qblue). An efficiency ratio of the used parameters was evaluated as the normalized ratio of FITC fluorescent level and viability. The purpose was to optimize the parameters for later transfection experiments and to get an overview of the influence of the different parameters. It was not intended to determine absolute transfection efficiencies.
A further parameter impacting the efficiency ratio is the particle size (Figure 3C). A higher efficiency ratio was reached with an increase of particle size. Up to a particle size of 150 nm no efficient permeabilization occurred. Using larger particle sizes, the efficiency ratio peaked at a radiant exposure of 60 mJ/cm2 for 200 nm and 60–80 mJ/cm2 for 250 nm particles before the efficiency ratio dropped due to laser induced cell damage.
Monitoring of the exposure effects on cell viability
The cell viability after performing permeabilization experiments at different radiant exposures and a fixed AuNP concentration of 11 μg/ml was followed up to 72 h. As presented in Figure 3D (left), the cell viability remained above 80% using radiant exposures up to 80 mJ/cm2. For a higher radiant exposure of 100 mJ/cm2 the cell viability strongly decreased to 40%.
The incubation of cells with AuNP at a concentration of 11 μg/ml for three hours without laser treatment did not show any pronounced effect on the viability for a time period of 72 h. Even the tripling of the AUNP incubation concentration to 33 μg/ml leads only to a slight decrease to 80-85% in cell viability. This negative effect on cell viability is likely to be caused by the residues of chloroauric acid used while particle manufacture.
Based on the presented results in Figure 3, the optimal parameter for an efficient cell permeabilization and tolerable cell loss is to a radiant exposure of 80 mJ/cm2, a particle size of 250 nm and an AuNP concentration of 11 μg/ml.
Nanoparticle mediated laser transfection
In order to evaluate a potential gene therapeutic approach, functional siRNAs were used in a proof of principle experiment using high cell numbers. For HMGA2 (high mobility group AT-hook 2) gene knock down experiments the canine HMGA2 overexpressing cell line CT1258  was transfected with four different anti-HMGA2 siRNAs complementary to the 3′-untranslated region of the HMGA2 mRNA and one non-sense scrambled siRNA. Due to the lack of reliable evaluated canine antibodies against the protein and thus potential unspecific cross reactions we opted for quantitative real-time PCR as detection method. This technique allows to measure the canine HMGA2 mRNA expression quantitatively.
The relative HMGA2 mRNA expression was analyzed 48 h after treatment via one step quantitative real time PCR (qRT-PCR) analysis (Figure 4G). The HMGA2 expression was quantified relative to the housekeeping gene Beta-actin (ACTB), the non-treated cells were used for calibration (reference value = 1). In all samples treated with HMGA2 specific siRNAs in combination with the laser manipulation suppression of HMGA2 could be observed. The highest suppression was induced by using the siRNA 1 and 2. For the siRNA 1 and 3, the gene knock down was significant compared to native cells (p-values < 0.05). In the control samples, no HMGA2 gene knock down could be observed. A slight increase was found for the scrambled siRNA, potentially resulting from off-target effects. No significant difference between the control samples and native cells was observed.
Characterization of the nanoparticle mediated membrane permeabilization mechanism
In this section we describe different experiments to address the mechanisms involved in membrane permeabilization focusing on the parameters allowing an efficient and gentle cell manipulation.
Near field and temperature related values for AuNP irradiated with 796 nm and 6.26 W/cm 2
Near field volume [nm3] (Imax/e2)
Average near field intensity [W/cm2]
Absorption efficiency Qabs
Particle temperature [K]
The evaluated enhancement factors and the applied intensity were used to calculate the near field intensity (Figure 5C (right y axis)). The values of the near field intensities of all particle sizes are below the threshold of an optical breakdown (LIOB) in water, which is 6×1012 W/cm2 for the used wavelength and pulse duration . The highest near field intensity is reached for 150 nm particles which is close to the LIOB threshold. Intensities below the LIOB threshold in the low density plasma regime can lead to nonlinear effects like multiphoton ionization and avalanche-ionization. This might lead to the permeabilization of the cell membrane [14,22,27].
Where E1 describes the threshold energy of a single pulse, N is the number of pulses and k the accumulation strength .
Threshold energy E 1 [μJ]
16.06 ± 0.7
5.88 ± 0.02
18.36 ± 0.7
5.88 ± 0.01
20.79 ± 0.7
5.55 ± 0.02
23.27 ± 0.7
5.00 ± 0.03
When laser radiation is absorbed by the electrons of the AuNP the energy is transferred from the electrons to the particle lattice due to electron phonon coupling within a time span of a few ps and the particle temperature increases [29,30]. The temperature of the electrons and the lattice can be calculated with a two-temperature model (Figure 5D) . The lattice temperature reaches the highest temperature of 726 K for 150 nm particles. This temperature is above the critical temperature (Tc) for phase transformation in water. For all other particles sizes this critical temperature is not reached. This reflects the different values for absorption efficiencies Qabs listed in Table 1.
Mean values ± SEM for the peak shift in the absorbance spectrum
Radiant exposure [mJ/cm 2 ]
The value of the peak shift for different particle sizes after laser radiation in dependence of the radiant exposure is shown in Figure 7B. The highest peak shift of 80 nm was measured for 150 nm particles and barley changes with increasing radiant exposures. Relating to the AuNP size of 250 nm used for transfection a peak shift occurred at radiant exposures ≥ 140 mJ/cm2. Furthermore, the amount of the peak shift for all radiant exposures is lower than for 150 nm particles which correlate with the calculated temperatures and near field enhancement in Figure 5.
In our study, we characterized the underlying mechanism and the potential of nanoparticle mediated cell membrane perforation in combination with fs-laser pulses as an alternative optical transfection method. Therefore the influencing parameters on the achieved perforation rate and cell viability were systematically determined and the successful transfection of cells with a fluorescent siRNA as well as the knock down of the oncogene HMGA2 in tumor cells with specific siRNAs was demonstrated. Furthermore, the passive binding of AuNP to the cell membrane was studied.
Multiphoton and scanning electron microscopy images show the localization of AuNP near the cell membrane. Depending on the incubation time of the AuNP, single particles or clusters are located near, or associated with, the membrane. After an incubation time of 3 h the AuNP are clearly visible near the cell membrane. Within this time the particles form clusters with enhanced scattering of the laser light proved by multiphoton microscopy . The agglomeration of particles after 3 hours is also visible in the ESEM images. This is in agreement with findings from Chithrani et al. who determined the uptake half-life at 2.24 h for 74 nm AuNP . Furthermore, they evaluated the uptake of the number of particles per cell and also showed that the number of particles per cell saturated after 5 h. In the present study a particle number of approx. 160 was estimated at the membrane of a single cell for an incubation concentration of 11 μg/ml. Baumgart et al.  counted per cell 90 ± 23 AuNP with a diameter of 100 nm at an incubation concentration of 8 μg/ml after an incubation time of 6 h using SEM images. Taking into account that in this work a higher particle concentration and a larger diameter was used (and therefore a faster sedimentation of the particles takes place) the results are in a very good agreement.
In addition, Chithrani et al. evaluated the number of AuNP per vesicle and found an average number of 3 AuNP per vesicle for 100 nm particles. In comparison to our SEM images (see Figure 2B) we assume that one 200 nm particle per vesicle get endocytosed by the cell. As bare AuNP are used, a serum protein corona is formed at the particles surface and no specific binding at the cell membrane is likely to occur. Therefore, we suggested the receptor-mediated endocytosis (RME) to be the acting uptake mechanism .
The initial mechanism of plasmon mediated cell membrane permeabilization is still a current matter in research. Depending on the parameters, different mechanisms and effects are assumed. These are thermal (“nanoheater effect”) [19,35], or near field enhancement effects (“nanolens effect”) [18,35]. In addition, the generation of a low density plasma induced by multiphoton ionization combined with thermal effects can possibly lead to membrane permeabilization [14,15]. For short laser pulses in the nanosecond-picosecond regime, where the energy is mainly absorbed by the particle, thermal effects could be the main mechanism for membrane permeabilization [36-38]. After AuNP heating, the water evaporates followed by a shockwave and forming a cavitation bubble around the exposed particles as reported by Pitsillides et al. and Zharov et al. and enabling membrane perforation [39,40]. Using fs laser pulses, nanocavitation bubbles can be formed by the induced field enhancement. This enhancement can lead to an optical breakdown near the particle and to the generation of a shockwave [18,36]. In this work, the evaluated intensities at the surface of single AuNP are near the threshold for an optical breakdown in the low density plasma regime. In existing studies, different concentrations of AuNP were required to achieve cell membrane perforation . Higher numbers of particles are necessary to manipulate the cells with fs laser pulses [22,41]. Due to the formation of AuNP clusters the near field is further enhanced in comparison to single particles. The neighboring particles interact via the scattered waves and due to plasmon coupling “hot spots” are formed [42,43]. Taking into account that the field enhancement is higher for AuNP clusters compared to single particles, the intensities could be above the optical breakdown threshold .
Our assumption herein, that clusters of AuNP at the cell membrane are necessary to induce a field enhancement by fs laser pulses which is high enough to perforate the cell membrane. This is supported by the presented microscopic images and the number of AuNP utilized in this and other studies using fs laser pulses for membrane perforation [22,41]. Within the performed experiments we showed the efficient and transient permeabilization of the cell membrane due to an expected enhancement of the near field at the AuNP clusters. Based on this and the evaluated simultaneous absorption of 5 photons in the pulse number dependent experiments (Figure 6) we understand the near field enhancement followed by the multiphoton ionization of the surrounding medium as the initial perforation mechanism.
Our studies on nanoparticle mediated fs laser cell perforation show, that this method is suitable for high throughput siRNA transfection with high efficiency and low cell toxicity. To establish this method as an alternative transfection technique, the manipulation of different cell types will be continued in further studies. However, due to the underlying physical mechanism the permeabilization should be cell type independent. Based on the mechanistic investigations, we assume that an enhancement of the near field occurs at AuNP clusters. This leads to the generation of a low density plasma with multiphoton ionization of the surrounding liquid, which in turn perforates the cell membrane. The uptake mechanism of extracellular molecules remains to be investigated in further experiments . The presented method is an alternative transfection method to deliver molecules into living cells being particularly well suited for standardized processes like high throughput or high content screening assays for fundamental and pharmaceutical research.
The canine pleomorphic mammary adenoma cell line ZMTH3  was cultured routinely in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Biochrom AG, Berlin, Germany). Rat granulosa cells (GFSHR-17) were cultivated in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 5% FCS, 1% penicillin/streptomycin (Biochrom AG, Berlin, Germany). The canine prostate adenocarcinoma cell line CT1258 was derived from an extremely aggressive canine prostate carcinoma  and cultured in Medium 199 (Life Technologies GmbH, Darmstadt, Germany) supplemented with 10% FCS and 2% penicillin/streptomycin (Biochrom AG, Berlin, Germany). The human ES cell line hES3 and the human iPSC line hCBiPS2  were cultured and expanded on irradiated mouse embryonic fibroblasts (MEF) in knockout DMEM supplemented with 20% knockout serum replacement, 1mM L-glutamine, 0.1mM β-mercaptoethanol, 1% nonessential amino acid stock (all from Life Technologies) and 10ng/ml bFGF (supplied by the Institute for Technical Chemistry, Leibniz University Hannover). One day before laser transfection cells were detached from the feeder layer by 0.2% collagenase IV (Life Technologies) followed by an incubation step with TrypLE (Life Technologies) for single-cell dissociation and plated onto Matrigel™ (BD Biosciences) coated dishes in MEF-conditioned medium.
The used automated setup for cell manipulation is operating with a fs amplifier laser system (Spitfire Pro, Newport Corporation, Irvine, USA). The generated laser pulses have a pulse duration of 120 fs at a fixed wavelength of 796 nm. The output power of the system is 2.1 W at a repetition rate of 5 kHz. To irradiate the biological tissue, the laser pulses were guided through an automatized attenuator consisting of a λ/2-plate and a polarizing beam splitter and reflected by two scanning mirrors (Litrack, JMLaser, Müller Elektronik, Spaiching, Germany). A convex lens with a focal length of 800 mm was used to focus the laser pulses onto the sample, located on the automatized stage (OptiScan, Prior, Jena, Germany), resulting in a spot diameter of 164 μm.
Prior to the laser cell manipulation experiments and to investigate the interaction of AuNP with the cell membrane, the cells were co-incubated with the AuNP at 37°C in a 5% CO2 atmosphere. The AuNP were chemically manufactured in presence of chloroauric acid (PGO, Kisker Biotech, Steinfurt, Germany). Uncoated AuNP of 80 nm, 150 nm, 200 nm, 250 nm were used.
Images were obtained to evaluate the incubation time for AuNP mediated cell permeabilization and the possibility of a passive binding of the particles. Briefly, granulosa cells were incubated with 150 nm gold particles and imaged after different incubation times. After a PBS wash, the cells were observed with a custom built multiphoton microscope which is based on a fs-laser system tunable from λ = 690 nm to 1040 nm (Chameleon ultra II, Coherent, Göttingen, Germany) . The images were recorded through a 100× oil immersion objective (Carl Zeiss AG, “Plan-Neofluar”, NA = 1.3) at an excitation wavelength of λexc = 700 nm.
Scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM)
To investigate the interaction of cells and AuNP images of ZMTH3 cells were generated after different times of co-incubation with 200 nm particles. The cells were washed after co-incubation with AuNP and fixed by adding a 4% paraformaldehyde (PFA) solution with 0.2% glutaraldehyde at 4°C. For ESEM imaging the cells were washed after 20 min with distilled water. For SEM, the cells were further treated at room temperature for 20 min with a 2% osmium tetroxide solution. Subsequently, the cells were washed 3 times with water for 5 min before incubation with different ethanol concentrations for 10 min each (30%, 50%, 70%, 90%, 95%, 95% and 3 × 100%). Before sputtering the cells with a 5 nm gold layer, the cells were dried for 30 min under laminar air flow conditions. For counting AuNP at the cell membrane after incubation, ImageJ was used . Values represent the mean of n = 6 images ± SEM.
Plate reader measurements
Simulation of the particle temperature and near field
For a deeper insight into the mechanisms involved in membrane permeabilization using fs laser pulses, the particle temperature and the near field were analyzed. The temperature of the AuNP during fs irradiation was calculated based on a two temperature model, employing data for the specific heat capacity of the electrons and the electron phonon coupling constant from Lin et al. . Temperature loss due to interaction with the surrounding medium was not considered due to the short timescales used. The field strength and intensity as well as the near field volume were simulated by the discrete dipole approximation, using the software DDSCAT [56,57]. A dipole separation of less than 3.5 nm was used for the largest sphere with a diameter of 250 nm. Modeling of the optical breakdown intensities in the near field was performed according to the Keldysh theory following the approach used by Vogel et al. [26,58]. The maximum intensity divided by the square of e was considered as near field volume and the enhancement in the modeling of the optical breakdown as well as the near field volume were averaged in the according area.
Particle spectra were monitored to evaluate a possible peakshift (as an indicator for a change in particle size/shape) of laser irradiated particles compare to untreated particles. Therefore an UV/Vis spectroscope (UV 1650-PC, Shimadzu, Duisburg, Germany) was used. The particles were diluted in culture media (RPMI as described before) without phenol red at a concentration of 50 μg/ml. Using a 96 well plate, the samples with a total volume of 200 μl per well were irradiated in a meander pattern.
In order to evaluate the transfection efficiency of the CT1258 cells, fluorescence microscopy was applied. 24 h before laser treatment, 1×104 cells were seeded in each well of a 24 well plate (PAA Laboratories, Cölbe, Germany). For siRNA transfection, 10 μM of a fluorescently labeled (AlexaFluor488) siRNA (Qiagen, Hilden, Germany) was added to the extracellular medium before laser treatment. The samples were treated with the optimized parameters as evaluated within the plate reader measurements. After laser treatment, the cells were incubated for 30 min followed by several washing steps until the background fluorescence from the fluorescent siRNA was eliminated. Three independent experiments in duplicates were performed on different days. Three images of each well were analyzed using Image J. By counting the cell nuclei (ca. 546 per image, stained with HOECHST33342) and transfected cells (Alexafluor488 siRNA positive cells) the transfection efficiency was determined. Propidium Iodide was used as an indicator for necrotic cells.
Flow cytometry analysis
Flow cytometric analysis was performed to evaluate the transfection efficiencies and the necrotic- and apoptotic rate. 24 h before laser treatment, 1.5×105 cells were seeded in each well of a 24 well plate. For siRNA transfection, 10 μM of a fluorescently labeled (AlexaFluor488) siRNA was added to the extracellular medium before laser treatment. The samples were treated with optimized parameters as evaluated within the plate reader measurements. Three hours after laser treatment the samples were prepared for flow cytometric analysis. Therefore, the cells were washed and trypsinized (TrypLE™, Life Technologies (LT), Darmstadt, Germany). A viability staining with Annexin V (V-PE-Cy5 Apoptosis Detection Kit, BioCat, Heidelberg, Germany) to detect the apoptotic cells, and with 1.5 μM Propidium Iodide (Invitrogen, Darmstadt, Germany) to identify necrotic cells, was performed. The positivity of siRNA transfected cells was determined by comparing the AlexaFluor488 fluorescence intensity to native cells, both measured in the FL1-H channel using a FACSCalibur flow cytometer (BD Bioscience, Heidelberg, Germany). Within the native cell population, a gate was set determining 98% of the native cells as non-transfected using the software Cell Quest (BD Bioscience, Heidelberg, Germany). This gate was subsequently applied on the siRNA transfected cell population resulting in the percentage of positive and non-transfected cells. To determine the ratio of apoptotic and necrotic cells within the siRNA transfected samples, the Annexin V and PI labelled cells were analyzed for PE-Cy5 fluorescence in the FL4-H channel and for PI in the FL2-H channel. Within the native cells a gate was set at which a cell population of 2% was identified as Annexin and PI positive and transferred to the sample with siRNA transfected cells to discriminate living from apoptotic and necrotic cells. For statistical analyses, the student’s t-test was used. The significance is given as * for p < 0.05, ** for p < 0.01 and *** for p < 0.001.
HMGA2 suppression analysis
Name and sequence of the used siRNAs for HMGA2 knock down
For PCR analysis total RNA was isolated according to the “NucleoSpin miRNA” protocol (Macherey & Nagel, Düren, Germany). Small and large RNAs were finally eluted in 30 μl nuclease free water. Total RNA concentration was measured with the Synergy 2 reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany).
Quantitative one step real-time PCR analysis
For the relative HMGA2 / ACTB quantification 25 ng total RNA were mixed with SYBR Green, HMGA2 or ACTB specific primers, nuclease free water (Qiagen, Hilden, Germany) and reverse transcriptase according to the “QuantiTect SYBR Green RT-PCR” protocol (Qiagen, Hilden, Germany). The fluorescence of each sample was analyzed in triplicates. As negative controls a non-template and a no-reverse transcriptase control were included. The experiments were performed using the Mastercycler ep realplex (Eppendorf AG, Hamburg, Germany). qRT-PCR conditions were as follows: 30 min at 50°C and 15 s at 95°C, followed by 40 cycles with 15 s at 94°C, 30s at 60°C and 30 s at 72°C. Finally a melting curve analysis was performed to verify specificity and identity of the qRT-PCR products according to the Eppendorf Mastercycler ep realplex instrument instructions. For the comparison of the relative gene expression levels based on the ∆∆CT method the gene expression level of the untreated CT1258 cells was used as calibrator (calibrator expression level was set as 1). Statistical analysis of the qRT-PCR results was done by using the software tool REST 2009, version 2.0.13. A p-value of ≤ 0.05 was considered as statistically significant.
The authors thank Regina Carlson for technical support in flow cytometry and the German Research Foundation DFG (within the Transregio 37 and the excellence cluster REBIRTH) for the financial support. We thank Ulrich Martin (Leibnitz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School) for providing the hES3 and hCBiPS2 cells.
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