The effect of mechanical conditioning

on collagen and collagen cross-link

mRNA expression

Internship

L.L.H. Both Identity number: 0567572

BMTE 08.24

April 2008

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Abstract Previous studies have demonstrated that the messenger ribonucleic acid (mRNA) expression of collagen type I is affected by mechanical loading in diverse cell types. These studies used cells cultured in a 2D- structure. In this study, 3D- cultured constructs are used to take cell- matrix interactions into account and therefore it is more physiologic. The goal of this study is to investigate the effect of mechanical conditioning on the collagen and collagen cross-link mRNA expression in Human Vena Saphena cells. The collagen architecture, including the amount of collagen type I and collagen cross- link influences the tissue properties. Therefore, collagen type I (Col I) and collagen cross-link enzyme lysine hydroxylase 2 (LH2) mRNA expressions are investigated in response to mechanical strain. The constructs are statically or dynamically loaded. Statically cultured constructs are attached to the bioflex plates by adding glue to the outer 5 mm of the strips and there is no external load applied. External cyclic strain with a frequency of 1 Hz at 4% strain is applied to the dynamically loaded constructs using the Flexercell straining system. To investigate the influence of mechanical loading two experiments are carried out. In these experiments, the constructs are respectively 1 or 6 day(s) statically cultured followed by a dynamic or static culture period of two days. The constructs, which were 1 day cultured before load was applied, showed no change in mRNA expression of statically and dynamically cultured constructs. The constructs with a culture time of 6 days before applying load showed a lower Col I mRNA expression and a higher LH2 mRNA expression in dynamically cultured constructs compared to statically cultured constructs after 48 hours. The foldchange of the 6 days cultured constructs before applying load also showed a significant lower Col I and a significant higher LH2 mRNA expression in dynamically cultured constructs compared to static constructs after 48 hours. Thus, dynamic loading seems to decrease the collagen type I and increase the collagen cross-link (LH2) mRNA expression. The effect of dynamic loading seems to be dependent on the duration of the culture time of the constructs.

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Table of contents 1 Introduction 4 2 Materials and methods 7

2.1. Cell culture 7 2.2. Construct fabrication and mechanical loading 7 2.3. Strain induced collagen and collagen cross- link expression 8

2.3.1. The influence of mechanical conditioning on collagen and collagen cross- link mRNA expression in 1 day cultured constructs 8

2.3.2. The influence of mechanical conditioning on collagen and collagen cross- link mRNA expression in 6 days cultured constructs 9

2.4. RNA isolation and cDNA synthesis 9 2.5. Real- time polymerase chain reaction 10 2.6. Data analysis 10

3 Results 12

3.1. The influence of mechanical conditioning on collagen and collagen cross- link mRNA expression in 1 day cultured constructs 12 3.2. The influence of mechanical conditioning on collagen and collagen cross- link mRNA expression in 6 days cultured constructs 13

4 Discussion and Conclusion 16 5 References 18 A Appendix 19

A.1. Culturing Human Vena Saphena Cells 19 A.1.1. Preparation of growth medium 19 A.1.2. Thawing the cells 19 A.1.3. Medium change 19 A.1.4. Subculturing the cells 19 A.1.5. Counting the cells 20

A.2. Preparation of scaffold and cell seeding 22 A.2.1. Preparation of reagents 22 A.2.2. Preparation of scaffold 22 A.2.3. Seeding of the scaffold 23 A.2.4. Culturing the constructs 24 A.2.5. Sacrificing the constructs 24

A.3. RNA isolation 25 A.3.1. Notes before starting 25 A.3.2. RNA isolation 25 A.3.3. Determination of the integrity and size distribution of the RNA 26 A.3.4. Quantification and purity of the RNA 27

A.4. cDNA synthesis 28 A.5. Real- time PCR 29

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1 Introduction Heart valve disease is a significant medical problem worldwide. It is difficult to cure and heart valve replacement is often required. One major drawback that all heart valve replacements have in common is the inability to grow, repair, and remodel after implantation in the body. Developing tissue engineered heart valves is a promising alternative to overcome these drawbacks. The concept of tissue engineering consists of autologous cells seeded on a biocompatible and biodegradable scaffold using fibrin gel as a cell carrier. These tissues are cultured in a bioreactor that mimics the physiological environment. It has been shown that seeding scaffolds using fibrin as a cell carrier offers a lot of advantages. It is a fast and efficiently seeding method and fibrin degrades fast over time [1]. In other studies [2], [3], it has been shown that a three dimensional fibrin structure can serve as a useful scaffold. Therefore, fibrin gels were tested by applying mechanical load but they were not able to bear this load. This is in correspondence with a previous study [3], where it was found that the initial stability was low. The combination of a biodegradable scaffold with the fibrin gel as a cell carrier was given as a good alternative. This is in correspondence to the findings of Mol et al. [1]. The obtained constructs are efficiently loaded using a Flexercell straining system. The structure and mechanical properties of tissues, such as tissue engineered heart valves, are dependent on the collagen architecture, including the amount of collagen, the collagen cross-link density and the orientation of the collagen fibers. Collagen fibers are bundles of collagen fibrils, made up of multiple collagen molecules (Figure 1.1). Three α- chains are twisted together into a collagen molecule. Collagen, like most proteins that are destined for transport to the extra cellular spaces for their function or activity, is produced initially as a larger precursor molecule called procollagen. Procollagen contains additional peptides at both ends that are unlike collagen. Once outside the cell, it undergoes propeptide cleavage and the additional peptides are removed. The telopeptides assemble into large arrays and form fibrils. It is stabilized by intermolecular cross- links.

Figure 1.1: The collagen structure. Left: Collagen fibers are made up of collagen fibers, which subsequently consist of multiple collagen molecules. The collagen molecules consist of three α-chains twisted together. Right: Procollagen is a precursor of collagen. Once outsise the cell, it undergoes propeptide cleavage and subsequently telopeptides assemble into large arrays and form fibrils. It is stabilized by intermolecular cross- links.

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The amount of matrix production of the cells can be determined but it takes a few weeks before the matrix is produced. Everything that is produced by the cell, like the matrix, comes first to expression (mRNA production) and after that it is synthesised. DNA is transcribed into mRNA in the nucleus of the cell. The mRNA is transported to the cytoplasm of the cell, where protein production proceeds (translation). The messenger ribonucleic acid (mRNA) expression is often determined because it is an early marker for the amount of matrix production and consequently it is not needed to culture the constructs for a few weeks but just a few hours/ days. In many studies it is demonstrated that cells are responsive to mechanical loading. It is shown that mechanical loading increases mRNA expression of collagen type I in several cell types [4 - 8]. An overview of these studies is given in table 1.1. In these various studies, different types of mechanical loading are applied to diverse cell types, which all resulted in an increased mRNA expression of collagen type I. Liu et all [4] have shown that the relative amount of mRNA of collagen type I remained stable when the strain was greater than 5%. This suggests that for each cell type optimal mechanical loading conditions exist. However these studies investigated the influence of mechanical loading on the collagen type I mRNA expression of cells cultured in a 2D- structure. In this study, 3D- cultured constructs are used to take cell- matrix interactions into account and therefore it is more physiologic than using 2D- cultured constructs. There are no earlier studies found in literature, which investigated the influence of mechanical loading on the collagen cross- link mRNA expression. Therefore this is a new and innovative study. Table 1.1: An overview of several studies at different cell types. It is shown that mechanical strain increases mRNA expression of collagen type I and that optimum loading condition for cells seems to exist. [4 – 8]

Cells Mechanical loading mRNA expression of collagen

type I

Human osteoblast- like cell line, SaOs-2 cells

5, 7.5, 10 and 12.5 % cyclic strain 0.5 Hz During 24 hours

5% strain: Increase > 5% strain: Stable

Human vascular smooth muscle cells

22% / 16% cyclic strain 1 Hz During 24 hours, measured after 12 hours

Increase

Rabbit chondrocytes 10% compressive strain 0.01, 0.05, 0.1 and 0.5 Hz Loading/unloading duration 2s/8s Measured after 24 hours

Increased frequency leads to increased expression

Porcine posterior tibial tendon fibroblasts

5% cyclic strain 0.5 Hz During 24 hours

Increase

Rat dermal fibroblasts 0, 4 and 8 % cyclic strain 1 Hz After 3 and 24 hours

Increased strain leads to increased expression

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The goal of this study is to investigate the effect of mechanical conditioning on the collagen and collagen cross-link mRNA expression. In earlier studies [4 - 8], it is shown that straining increases the expression of collagen type I. It is also known that mechanical conditioning increases the mechanical properties of tissues. The collagen architecture, including the amount of collagen type I and collagen cross- link influences the tissue properties. Therefore the collagen type I (Col I) and collagen cross-link enzyme lysine hydroxylase 2 (LH2) mRNA expressions are investigated in response to mechanical strain. LH2 is a precursor for the formation of cross-links which provide collagen its tensile strength. Human Vena Saphena cells seeded on a scaffold using fibrin gel as a cell carrier are used as a model system for cardiovascular tissues such as heart valves. In this study, the constructs are statically and dynamically loaded using a FlexCell straining system. Statically cultured constructs are attached to the bioflex plates by adding glue to the outer 5 mm of the strips and there is no external load applied. External cyclic strain is applied to the dynamically loaded constructs. To investigate the influence of mechanical loading two experiments are carried out. In these experiments, the constructs are respectively 1 or 6 day(s) statically cultured followed by a dynamic or static culture period of two days. The effect of the different loading conditions on gene expressions are investigated using real- time or quantitative polymerase chain reaction (qPCR). It is hypothesized that mechanical strain will increase collagen and collagen cross-link expression.

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2 Materials and methods

2.1. Cell culture

Human Vena Saphena cells (HVSC’s) were cultured in culture medium, containing DMEM Advanced, 10% fetal bovine serum (FBS), 1% glutamax and 0.1% Gentamycin. Cells were cultured at 37˚C in an incubator. Culture medium was changed every three to four days. After cells reached 80- 90% confluence, they were released with trypsin and further subcultured. The protocol is given in Appendix A.1.

2.2. Construct fabrication and mechanical loading

Rectangular strips (33 × 5 × 1 mm) of polyglycolic acid (PGA) scaffold were coated with 1% w/v poly-4-hydroxybutyrate (P4HB) dissolved in tetrahydrofuran (THF). The strips were dried overnight in a vacuum stove to vaporize all the remaining THF and attached to the flexible bottom of the bioflex plate by adding glue to the outer 5 mm of the strips (figure 2.1). The scaffolds were placed in 70% EtOH for at least 5 hours to sterilize it and dried overnight. 24 Hours before seeding cells, the scaffolds were washed with PBS, followed by adding TE medium, consisting of DMEM Advanced (500ml), 10% fetal bovine serum (FBS), 1% glutamax,0.3% Gentamycin and L-ascorbic acid 2- Phosphate (130 mg). The cells were released, counted and resuspended in the thrombine solution. Subsequently the fibrinogen was added and the cells were seeded on the scaffold at a density of approximately 1.5 million cells per 100 mm3. Before TE medium was added to the constructs, it was placed in the incubator for 30- 45 minutes to let the fibrin gel further polymerize. The constructs were cultured at 37˚C in an incubator and mechanical loading was applied using a Flexercell straining system. A schematic representation of the flexercell straining system is given in figure 2.2. The construct is attached to the flexible membrane of the bioflex well, which is placed on a loading post. A small part of the flexible membrane is not covered by the loading post. By applying a vacuum to the device, this part of the flexible membrane is stretched down and consequently the constructs will undergo strain. The protocol for construct fabrication is given in appendix A.2.

Figure 2.2: Schematic respresentation of the Flexercell straining system. The construct is attached to the flexible membrane of the bioflex well, which is placed on a loading post. The flexible membrane is stretched down by applying a vacuum to the device and consequently the constructs will undergo strain.

Bioflex well

Loading post

medium

vacuum

Flexible membrane Scaffold and cells

Figure 2.1: PGA/P4HB scaffold attached to the flexible bottom of the bioflex plate by adding glue to the outer 5 mm of the strips.

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2.3. Strain induced collagen and collagen cross- link expression

To study the effect of mechanical loading on Col I and LH2 expressions, constructs were cultured statically or dynamically (4% cyclic strain, 1Hz). Statically cultured constructs are attached to the bioflex plates as described in section 2.2 and there is no external load applied. External cyclic strain is applied to the dynamically loaded constructs by using the Flexercell straining system as described in section 2.2. Two experiments are carried out to investigate the influence of mechanical loading. In these experiments, the constructs are respectively 1 or 6 day(s) statically cultured followed by a dynamic or static culture period of two days as further described in section 2.3.1 and 2.3.2. At the end of the culture period, the constructs were removed from the bioflex plates, cut into two pieces, separately frozen in nitrogen and stored at -80˚C until analysis.

2.3.1. The influence of mechanical conditioning on collagen and collagen cross- link mRNA expression in 1 day cultured constructs

The used cells were cultured in T150 flasks until the 8th passage. Constructs were statically cultured for 1 day, followed by a dynamic or static culture period of 2 days. A schematic representation of the culture periods is given in figure 2.3. During the last 2 days, constructs were sacrificed for analysis after 2, 4, 6, 10, 24 and 48 hours. The numbers of samples of the statically and dynamically cultured constructs at these time points, when the constructs are sacrificed, are given in table 2.1.

Figure 2.3: Schematic representation of the culture periods of the 1 day cultured constructs before applying load. Constructs were statically cultured for 1 day, followed by a dynamic or static culture period of 2 days. During the last 2 days, constructs were sacrificed for analysis after 2, 4, 6, 10, 24 and 48 hours.

Table 2.1: The number of samples of static and dynamic constructs at different culture times after 1 day of static culture.

Culture time after 1 day statically

culture time [Hours]

2 4 6 10 24 48

Static 1 1 2 2 2 1

Dynamic 2 2 2 2 2 2

Static culture period of 1 day 2 4 6 10 24 48 Hours

2 4 6 10 24 48 Hours

Static culture period of 2 days

Dynamic culture period of 2 days

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2.3.2. The influence of mechanical conditioning on collagen and collagen cross- link mRNA expression in 6 days cultured constructs

Cells were cultured until passage 7 in roller bottles. The constructs were cultured statically for 6 days, followed by a static or dynamic culture period of 2 days. A schematic representation of the culture periods is given in figure 2.4. During the last 2 days, the constructs were sacrificed for analysis after 0, 3, 6, 33 and 48 hours. The numbers of samples of static and dynamic constructs after different culture times, when the constructs are sacrificed, are given in table 2.2.

Figure 2.4: Schematic representation of the culture periods of the 6 days cultured constructs before applying load. Constructs were statically cultured for 6 days, followed by a dynamic or static culture period of 2 days. During the last 2 days, constructs were sacrificed for analysis after 0, 3, 6, 33 and 48 Hours.

Table 2.2: The number of samples of static and dynamic constructs at different culture times after 6 days of static culture.

Culture time after 6 days statically

culture time [Hours]

0 3 6 33 48

Static 2 2 2 1 1

Dynamic 2 2 2 2 1

2.4. RNA isolation and cDNA synthesis

Total cellular RNA was extracted using Qiagen RNeasy Kit, according to the protocol in Appendix A.3. Frozen samples were crushed and homogenized in RLT buffer. EtOH was added and the lysate was transferred to the column followed by a few washing steps. Finally, RNAse- free water was added to the column, to elute the RNA. The integrity and size distribution of the isolated RNA was determined using gel electrophoresis. Samples were loaded on a 1% agarose gel and visualized with UV- light. When the 28 S band was twice as thick as the 18 S band and no other sizes of RNA were visible, the RNA was integer. This can be seen in figure 2.4. The amount and purity of the isolated RNA was determined by measuring the absorptions at 260 and 280 nm. The ratio between these absorptions should be above 1.6. Above 1.8 indicates a pure sample. The RNA concentration was calculated.

Static culture period of 6 days

0 3 6 33 48 Hours

Static culture period of 2 days

Dynamic culture period of 2 days

0 3 6 33 48 Hours

Figure 2.4 RNA visualized on a 1% agarose gel using UV- light. RNA is integer when the 28 S band is twice as thick as the 18 S band and no other sizes of RNA are visible.

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(Equation 2.1)

(Equation 2.2)

Complementary deoxyribonucleic acid (cDNA) was synthesized of 500 ng isolated RNA, according to the protocol in appendix A.4. First, the reverse transcriptase mix consisted of 2.5µl random primers, 2.5µl 5mM dNTPs and 5.0 µl first strand buffer was added to the RNA. Subsequently, the samples were 6 minutes incubated at 72˚C and 5 minutes at 37˚C. The enzyme mix consisted of 1.0 µl ddH2O, 2.5 µl DTT and 0.5 µl M-MLV was added, followed by 1 hour incubation time at 37˚C and 5 minutes at 95˚C. cDNA was stored at 4˚C until qPCR was carried out.

2.5. Real- time polymerase chain reaction

qPCR was performed in 15 µl reaction volume, consisted of 7.5 µl SYBR green supermix, 6.5 µl ddH2O, 0.375 µl of each primer (20 µM) and 1.0 µl cDNA. The thermal cycling program for qPCR was: 3 minutes at 95˚C; 40 cycles of 20 seconds at 95˚C, 20 seconds at 60˚C and 30 seconds at 72˚C; 1 minute at 95; 1 minute at 65˚C; and followed by 61 cycles of 10 seconds starting at 65˚C and gradually increase till 95˚C to obtain a melting curve. The temperature corresponding to the top of the melting curve is related to the length of the PCR product and is often unique for every target gene. Therefore, this is an indication for contamination. Figure 2.2 shows the amplification curves of the qPCR measurement of two different samples. The threshold cycle (denoted by Ct) represents the cycle number at which the curve crosses the threshold line. The threshold is positioned at the y-value where the curve becomes linear. The lower the Ct value, the higher the original mRNA amount of the target gene in the sample Thus, in this figure, sample 1 contains more target gene mRNA than sample 2.

Figure 2.2: Amplification curve of the real-time PCR measurement.

Each sample was measured in duplo and Ct values were further analyzed as described in section 2.6. The protocol is given in Appendix A.5. The qPCR products were visualized on a 2% agarose gel.

2.6. Data analysis

The data obtained from qPCR was analyzed by determining the normalized amount of mRNA and the foldchange. The normalized amount of mRNA (N) is calculated with equation 2.1:

nCT

nN∆−

= 2

With: enreferencegnn CtCtCt −=∆

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(Equation 2.3)

(Equation 2.4)

(Equation 2.5)

In this study, the amount of Col I and LH2 mRNA is normalized to the amount of the reference genes: GAPDH and/ or β-actin. The foldchange between two samples is calculated with equation 2.3:

nCTFoldchange

∆∆−= 2

With: controlenn CtCtCt ∆−∆=∆∆

In this study, nCt∆∆ is the difference in Ct value between a static and a dynamic

cultured sample at the same moment. Foldchange values larger than 1 indicate an increased expression. A decreased expression is indicated by values smaller than 1. The results are significant by foldchanges larger than 2 or smaller than 0.5. During data analysis, the following calculations were made:

− Ct values were averaged over the duplo measurements in qPCR.

− After sacrificing the scaffolds for analysis, the scaffolds were divided in two

parts. The average over both nCt∆ values is taken for further calculations. The

standard deviation is calculated over these samples with equation 2.5.

)1(

)( 2

11

2

−

−

=∑∑ =

=

NN

xxNN

i i

N

i

i

σ

− The numbers of samples at each data point are shown in table 2.1 and 2.2. Some samples are left out because there was not enough amount of RNA for cDNA synthesis or it was not integer. Therefore some results are based on 1 sample.

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3 Results

3.1. The influence of mechanical conditioning on collagen and collagen cross- link mRNA expression in 1 day cultured constructs

The amounts of collagen type I and collagen cross-link (LH2) mRNA normalized to GAPDH of dynamically and statically cultured constructs are plotted in Figure 3.1. Both dynamically and statically cultured constructs showed no difference in Col I and LH2 mRNA expression in time. The foldchange of Col I and LH2 expressions between dynamically and statically cultured constructs are shown in Figure 3.2. Foldchanges of both Col I and LH2 mRNA expressions did not differ significantly between dynamically and statically cultured constructs.

Figure 3.1: mRNA amount of Col I and LH2 normalized to GAPDH amount for dynamically and statically cultured constructs. There is no difference in Col I and LH2 mRNA expression of dynamically and statically cultured constructs in time. Data is represented as mean ± standard deviation.

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Figure 3.2: Foldchange of Col I and LH2 mRNA expression between dynamically and statically cultured construct. There is no significant difference between dynamically and statically cultured constructs. Foldchanges larger than 2 or smaller than 0.5 are considered to be significant.

3.2. The influence of mechanical conditioning on collagen and collagen cross- link mRNA expression in 6 days cultured constructs

The amounts of collagen type I and collagen cross-link enzyme (LH2) mRNA normalized to GAPDH and β-actin of dynamically and statically cultured constructs are respectively shown in figure 3.3 and 3.4. Figure 3.5 shows the foldchange of Col I and LH2 mRNA expressions of dynamically cultured constructs compared to statically cultured constructs. Dynamically cultured constructs showed a significant lower Col I expression normalized to both GAPDH and β- actin compared to statically cultured constructs after 48 hours. Furthermore, the LH2 mRNA expression of dynamically cultured constructs compared to statically cultured constructs normalized to both GAPDH and β- actin was significant higher after 48 hours. There is no significant difference in the foldchange of both Col I and LH2 mRNA expressions between dynamically and statically cultured constructs untill 48 hours.

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Figure 3.3: mRNA amount of Col I and LH2 normalized to GAPDH amount for dynamically cultured constructs compared to static constructs. After 48 hours, the normalized amount of LH2 was higher in dynamically cultured constructs compared to static constructs. The normalized amount of Col I mRNA was lower in dynamically cultured constructs compared tot static constructs after 48 hours. Data is represented as mean ± standard deviation

Figure 3.4: mRNA amount of Col I and LH2 normalized to β-actin amount for dynamically cultured constructs compared to static constructs. After 48 hours, the normalized amount of LH2 was higher in dynamically cultured constructs compared to static constructs. The normalized amount of Col I mRNA was lower in dynamically cultured constructs compared tot static constructs after 48 hours. Data are represented as mean ± standard deviation.

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Figure 3.5: Left: Foldchange of both Col I and LH2 mRNAexpression normalized to GAPDH expression. Right: Foldchange of Col I and LH2 expression normalized to β- actin expression. After 48 hours, dynamically cultured constructs showed a significant lower Col I expression and a significant higher LH2 expression normalized to both GAPDH and β- actin. Foldchanges larger than 2 or smaller than 0.5 are considered to be significant.

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4 Discussion and Conclusion The effect of mechanical loading on the collagen and collagen cross-link mRNA expression was investigated. Therefore constructs were 1 day statically cultured, followed by a static or dynamic culture period of 2 days. There was no difference in Col I and LH2 mRNA expression between statically and dynamically cultured constructs in time (figure 3.1). This suggests that probably other factors, like the presence of the scaffold may influence the amount of mRNA expression of the cells in the constructs. This is because it might be that less strain is felt by the cells due to the presence of the scaffold. Therefore a second experiment was carried out with a longer tissue culture time before mechanical load was applied. Constructs were 6 days statically cultured, followed by a static or dynamic culture period of 2 days. The dynamically cultured constructs showed a lower Col I mRNA expression and a higher LH2 mRNA expression compared to statically cultured constructs after 48 hours (figure 3.3 and 3.4). 48 Hours after a statically culture period of 6 days, the foldchange showed a significant lower Col I and a significant higher LH2 mRNA expression in dynamically cultured constructs compared to statically cultured constructs (figure 3.5). Assuming that the entire gene expression of cells is translated into protein expression, cells are stimulated to form collagen cross-links after 6 days of static culture followed by 2 days of dynamic culture. This was not the case in constructs which have had a culture time of 1 day. These results might suggest that the load applied to the cells increases as a function of culture time due to the degradation of the scaffold in time. In an earlier study, it is shown that the mechanical properties of the PGA/P4HB scaffold dramatically decrease as a function of culture time [9]. Normally, without loading the scaffold starts to degrade after 10 to 14 days and will consequently lose its mechanical integrity. But it is also might be that mechanical loading supports the degradation of the scaffold. Since the scaffold degrades in time, the load becomes less borne by the scaffold. Therefore, the applied load to the cells may increase when the scaffold breaks down. To survive this load, the cells have to adapt quickly to the new circumstances, by probably making more collagen cross-links. The collagen cross- links provide collagen its tensile strength and therefore it might be the most efficient manner to survive the load in the short term. Consequently, the cells may temporary lower their collagen production to compensate for the increased collagen cross- link production. This may explain the higher collagen cross-link and lower collagen mRNA expression in constructs after 6 days statically culture followed by 2 days dynamically culture. To obtain a tissue, like a heart valve with the optimal structure and mechanical properties, the loading conditions have to be ideal. It is shown it previous studies that the Flexercell straining system is a valuable tool to apply global strains to tissue [10]. However, local strains which are important for matrix production, including collagen and collagen cross-links by cells can differ between neighbouring cells due to the inhomogeneous scaffold properties. Thus, correlations between global strain and local tissue remodelling can not be made. Therefore, multi level computational model, combined with local measurements of strains and matrix architecture are required to correlate local and global strains to tissue properties [11]. It is also important to integrate the degradation of the scaffold in the model because it probably has a lot of influence on the local strain felt by cells. Therefore, the degradation of the scaffold needs to be investigated in future studies. The multi level computational model may be used to optimize the loading conditions for cells and consequently to obtain tissues

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with properties comparable to the tissues in the human body. In this study, the strain felt by the cells might have been increased in time due to the degradation of the scaffold. Therefore, multi level computational model may be used to calculate the global strain which should be applied to the constructs to maintain the ideal local strain. The number of samples should also be increased in future studies. In conclusion, dynamic loading seems to decrease the collagen type I and increase the collagen cross-link (LH2) mRNA expression. The effect of dynamic loading seems to be dependent on the duration of the culture time of the constructs.

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5 References [1] A. Mol, M. I. v. Lieshout, C.G. Dam-de Veen, S. Neuenschwander, S.P.

Hoerstrup, F.P.T. Baaijens and C.V.C. Bouten, Fibrin as a cell carrier in cardiovascular tissue engineering applications, Biomaterials 26 (2005), pp. 3113-3121

[2] Q. Ye, G. Zünd, P. Benedikt, S. Jockenhoevel, S.P. Hoerstrup, S. Sakyama, J.A. Hubbell and M. Turina, Fibrin gel as a three dimensional matrix in cardiovascular tissue engineering, Eur J Cardio-thorac Surg 17 (2000), pp. 587-591

[3] S. Jockenhoevel, G. Zund, S.P. Hoerstrup, K. Chalabi, J.S. Sachweh, L. Demircan, B.J. Messmer and M. Turina, Fibrin gel – advantages of a new scaffold in cardiovascular tissue engineering, Eur J Cardio-thorac Surg 19 (2001), pp. 414-430

[4] X. Liu, X. Zhang and Z.P. Luo, Strain- related collagen gene expression in human osteoblast- like cells, Cell tissue Res 322 (2005), pp. 331-334

[5] A.G Stanley, H. Patel, A.L Knight and B. Williams, Mechanical strain- induced human vascular matrix synthesis: the role of angiotensin II, J Renin Angiotensin Aldosterone System 1 (2000), pp. 32-35

[6] J. Xie, Z. Han, S.H. Kim, Y.H. Kim and T. Matsuda, Mechanical loading-dependence of mRNA expressions of extracellular matrices of chondrocytes inoculated into elastomeric microporous poly(L-lactide-co-ε-caprolactone) scaffold, Tissue Engineering 13 (2007), Number 1

[7] C.H Chen, J.V. Marymont, M.H. Huang, M. Geyer, Z.P. Luo, X. Liu, Mechanical strain promotes fibroblast gene expression in presence of corticosteroid, Connective Tissue Research, 48 (2007), pp. 65-69

[8] W. A. Loesberg, X. F. Walboomers, J.J.W.A. van Loon, J.A. Jansen, The effect of combined cyclic mechanical stretching and microgrooved surface topography on the behavior of fibroblasts, J. of Biomedical Materials research Part A, Vol. 75A (2005), Issue 3, pp.723-732

[9] L. Kouda, E. C.M. Vaz, A. Mol, F.P.T. Baaijens and C.V.C. Bouten, Effect of biomimetic conditions on mechanical and structural integrity of PGA/P4HB and electrospun PCL scaffolds, J Mater Sci: Mater Med 19 (2008), pp.1137–1144

[10] R.A. Boerboom., M.P. Rubbens, N.J. Driessen, C.V. Bouten, and F.P. Baaijens, Effect of strain magnitude on the tissue properties of engineered cardiovascular constructs, Ann. Biomed. Eng 36 (2008), pp.244-253

[11] R.G.M. Breuls, B.G. Sengers, C.W.J Oomens, C.V.C Bouten and F.P.T. Baaijens, Predicting local cell deformations in engineered tissue constructs: a multilevel finite element approach, J Biomech Eng 124(2002), pp.198-207

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A Appendix

A.1. Culturing Human Vena Saphena Cells

A.1.1. Preparation of growth medium

− DMEM Advanced (500 ml)

− 10% FBS (50 ml)

− 1% Glutamax (5 ml)

− 0.1% Gentamycin (0.5 ml)

A.1.2. Thawing the cells

Normally the cells are frozen in an amount of ~2 X 106 cells per vial. This is a good amount to start with in a T150 flask. The passage that is on the vial is the passage at which they were frozen, so add a passage when you set up the cells.

1 Take a vial out of the liquid nitrogen 2 Warm up between your hands 3 Carefully open the vial to let N2 escape 4 Put 10 ml PBS in a 50 ml centrifuge tube 5 Transfer the contents of the vial (2 X 106 cells) to the centrifuge tube with the

PBS 6 Rinse the vial with PBS and add to the centrifuge tube 7 Centrifuge 7 min at 1000 rpm (or 5 min at 1500 rpm) 8 Discard the supernatant and resuspend the cells in 10 ml medium 9 Transfer the cells to a T150 flask at an amount of 0.5 X 106 cells per vial 10 Rinse the centrifuge tube with another 15 ml of medium and add this to the

T150 flask 11 Mix the cells with the medium gently and place the flask in the incubator

(always check the water level in the incubator!)

A.1.3. Medium change

The medium should be changed every three to four days. Most convenient is to do this every Tuesday and Friday or every Monday and Thursday.

1 Discard the medium 2 Rinse the cells once with PBS 3 Add new medium up to 25 ml per flask

A.1.4. Subculturing the cells

First subculturing after thawing:

It will take about 7-10 days to grow confluent. Subculture the cells (you will have about 5-6 X 106 cells) at a seeding density of 1.0 X 106 cells per new flask.

Further subculturing:

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It will take them about 7-14 days before they grow 80-90% confluent. Subculture them (you will then have about 5 X 106 cells per flask) at a seeding density of 1.0 X 106 cells per new flask.

1 Discard the medium 2 Rinse the cells with PBS 3 Add trypsin (2.5 ml per T150 flask) and distribute evenly over the bottom of

the flask 4 Place the vial in the incubator for 7 minutes 5 Check whether all the cells are rounded up and de-attached. If not place the

flask back into the incubator for a few more minutes. 6 Add 5 ml of medium to the flask and mix the medium with the cells and the

trypsin 7 Transfer the medium to a centrifuge tube 8 Add 10 ml of PBS to the flask and pipette up and down to the walls of the

flask to get all the leftover cells transferred into the centrifuge tube 9 Check the flask microscopically for leftover cells. If there are still many cells

in there rinse the flask again with PBS and add this to the centrifuge tube 10 Centrifuge 7 minutes at 1000 rpm (or 5 min at 1500 rpm) 11 Discard the supernatant and resuspend the cells in 1 ml medium for counting

(described separately) 12 Count the cells 13 Calculate the amount of new flasks needed to get a seeding density of 0.7 – 1.0

X 106 cells per new flask 14 Add medium to the cells and resuspend 15 Divide the cells over the desired amount of new flasks 16 Fill each flask up to 25 ml with medium and place the flasks in the incubator

Note: You can continu the subculturing procedure up to passage 8-9, after that

passage they will not be the same cells anymore as you can notice in a changed morphology and a different growth speed.

Note: Best is to count the cells every time you transfer them to keep track of their

growing speed. Prepare a growth curve and check whether the speed does not change over the culture period.

A.1.5. Counting the cells

Important for the counting procedure is to think of the amount of cells you expect to have in your flasks to calculate the right dilution factor and to prevent that you will have too less or too many cells in your counting window. Always try to have 100-150 cells in your counting window for a reliable counting.

1 Prepare the counting window: clean, put the cover glass on top and check for dust

2 After resuspending the cells in 1 ml of medium, take out 50 µl of the suspension and transfer this to a small centrifuge tube, which will be your counting tube

3 Calculate the dilution factor for counting: For example if you would expect 15 X 106 cells, you should dilute the cell suspension 10 times to get 150 cells in

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your counting window: 15 X 106 (expected amount) / 10.000 (factor of the Neubauer counting window) / 150 (desired amount of cells to count)

4 Add PBS to the counting tube to obtain the desired dilution and resususpend the cells in the PBS

5 Put 11µl of the counting solution at each side of the counting window 6 Count the cells and check viability (living cells appear as round cells with a

white edge, dead cells are dark and have irregular edges 7 Calculate the amount of cells in your suspension: 8 Counted cells X dilution factor X 10.000 9 Discard the counting solution

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A.2. Preparation of scaffold and cell seeding

A.2.1. Preparation of reagents

The medium used for tissue engineering differs from the medium used for cell culture. The amount of antibiotics used is a bit larger to prevent infections and L-ascorbic acid 2-phosphate is added to stimulate matrix formation.

Culture medium:

− DMEM Advanced (500ml)

− 10% FBS (50ml)

− 1% glutamax (5ml)

− 0.1% gentamycin (0.5ml)

TE medium:

− DMEM Advanced (500ml)

− 10% FBS (50ml)

− 1% glutamax (5ml)

− 0.3% gentamycin (1.5ml)

− L-ascorbic acid 2-phosphate, Sigma (130mg): non sterile! L-ascorbic acid 2-phospate has to be solved in DMEM Advanced and sterile filtered: Therefore, add 15ml of DMEM Advanced to a 50ml tube, warm this for several minutes in the warm water bath (in warm medium the ascorbic acid will more easily dissolve), add 130mg L-ascorbic acid 2-phosphate, put it back in the warm water bath. After about 10 minutes it will be dissolved, sterile filter it into the rest of DMEM. Then add the rest of the components to the medium

P4HB solution:

Dissolve 0.3 gram P4HB in 30 ml THF to obtain a 1% w/v solution.

A.2.2. Preparation of scaffold

1 Cut rectangular strips of approx. 33*5mm out of the PGA sheet 2 Coat the PGA strips with P4HB by dipping the scaffold into the P4HB

solution 3 Leave the scaffold to dry on a glass plate in the cabinet 4 Let the scaffolds further dry in the vacuum stove in a centrifuge tube with

holes in the lid overnight. 5 The scaffold strips can then be mounted in the bioflex plates Put a droplet of

the silicone gel on either sides of the well. Put also some silicone gel on both sides of the scaffold. Press the scaffold strips firmly into the silicone gel droplets.

6 Let the scaffold dry overnight (no vacuum). 7 Fill the wells with 70% alcohol and leave it for at least 3h (work in the LAF

cabinet) 8 Remove the alcohol and let the wells dry in the LAF cabinet 9 Rinse the wells thoroughly with PBS and fill the wells with TE medium and

place in an incubator overnight.

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A.2.3. Seeding of the scaffold

1 Prepare the thrombin solution: weight about 2.0 mg of thrombin and transfer to a centrifuge tube.

2 Add an amount of TE medium to obtain a concentration of 10 IU/ml. Look at the thrombin pot to see how much IU go into 1mg. If, for example, it says 1mg contains 34.7 IU, you will need a concentration of 0.288mg thrombin per ml medium.

3 Shake the solution and put on ice for about 10 minutes 4 Sterile filter the solution with the syringe and the sterile filter. 5 Store the sterile solution on ice until use

6 Prepare the fibrinogen solution: weight about 50mg of fibrinogen and transfer

to a centrifuge tube. 7 Add an amount of TE medium to obtain a concentration of 10mg actual

protein per ml medium. If, for example, the amount of actual protein is 80% you will need 12.5mg/ml medium to obtain a concentration of 10mg actual protein/ml medium.

8 Mix (gently shake) the solution for a couple of minutes until (almost all) the fibrinogen is dissolved and sterile filter this. Sometimes you will need multiple sterile filters for this.

9 Store the sterile solution on ice until use.

10 Before you start, calculate the amount of cells you need:

11 Per strip you will need about 150µl of cell suspension, equalling the volume of your strip. The seeding density can be varied but 10.000 cells/mm3 = 10*106 cells/ml normally gives good results. In this case you will need 1.5*106 cells/strip.

12 Rinse the desired amount of flasks or rollerbottles (RB) with cells with PBS 13 Add 2.5ml (150 flask) or 10ml (RB) of trypsin and distribute evenly over the

bottom/surface. 14 Place the flasks/RB for 5-10 minutes in the incubator. 15 Check whether all the cells are rounded up and de-attached. If not place the

flasks/RB back into the incubator for a few more minutes. 16 Add culture medium to the flasks/RB and mix the medium with the cells and

trypsin 17 Transfer the medium with cells to the centrifuge tube 18 Rinse the flasks/RB with PBS and add to the centrifuge tube 19 Centrifuge 7 minutes at 1000rpm 20 Discard the supernatant and resuspend the cells in 1ml medium for counting 21 Count the cells (as described in the Appendix A.1.A.1.5) 22 Resuspend the cells in 10ml PBS 23 Centrifuge them again at 7 minutes at 1000 rpm 24 Discard the supernatant and resuspend the cells in the sterile thrombin solution

so that you obtain a concentration of 20*106 cells/ ml thrombin (this will result in a concentration of 10*106 cell / ml fibrin)

25 Remove the medium from the well and aspirate the remaining medium from the strip. It is important that the scaffold is nearly dry before seeding.

26 To seed 2 strips, add 150µl of the thrombin-cell solution to a 5ml tube, take

150µl of sterile fibrinogen in the pipet tip and put the pipet to 400µl

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27 When fibrinogen is added to the thrombin, the solution starts to gel very quickly. Therefore you need to adjust the volume of the pipet before you start

mixing. The total volume of fibrinogen and thrombin in this case is 300µl, but the cells have a volume as well. To not loose any of the cells, take a larger volume

28 Mix the cell/thrombin solution gently with the fibrinogen for several times and then immediately pipet the mixture at several spots of the 2 strips.

29 After seeding all of the strips in the bioflex plate, place the scaffold with cells in the incubator for about 30 minutes to let the fibrin gel further polymerize.

30 Carefully add TE medium to the constructs and place them back into the incubator.

A.2.4. Culturing the constructs

Change the medium every 2-3 days. Do not rinse with PBS. Preferably, put your 6-wells plates in the incubator on a shaking table to allow mixing of the medium

− Remove Medium

− Add new TE Medium to the constructs

A.2.5. Sacrificing the constructs

After the desired amount of culture days, the scaffolds need to be sacrificed to determine collagen and collagen mRNA expression.

− Use a sterilised knife to sacrifice the construct

− Put it on a clean sheet of glass and cut it in two parts

− Transfer the pieces to a vial

− Put the vials in the nitrogen

− Store them in the -80˚C fridge

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A.3. RNA isolation

A.3.1. Notes before starting

RNA is highly unstable and very sensitive to degradation by specific enzymes, the RNases. It is therefore important that you work neatly and fast. Change your gloves regularly and keep your samples frozen and on ice as long as possible. When you want to isolate multiple samples in one run, you can keep your samples for some time on ice after the cell lysis and homogenisation procedure. The lysis buffer contains RNase inhibitors that protect your samples from degradation. Before starting the isolation procedure, put all samples at room temperature for 15 minutes (the binding capacity of the column is improved at 20˚C). The RNA isolation procedure should be performed quickly and at constant temperature (do not put your samples on ice in between the steps). Therefore, it is important to first collect all your lysated and homogenized samples before running them through the columns.

A.3.2. RNA isolation

1 Crush the frozen samples by shaking them 3 times for 30 seconds at high speed

2 Disrupt cells by addition of Buffer RLT and homogenize 3 Add the appropriate volume of Buffer RLT (Table A.1) and mix well by

vortexing or pipetting.

Table A.1: Amount of RLT buffer per amount of cells

Number of cells Buffer RLT

< 5 × 106 350 µl

5 × 106- 1 × 107 600 µl

4 Add 1 volume (usually 350 µl or 600 µl) of 70% ethanol to the homogenized

lysate, and mix well by pipetting. Do not centrifuge. If some lysate is lost during homogenization, adjust volume of ethanol accordingly. Continue directly with the next step.

5 Note: Visible precipitates may form after the addition of ethanol when preparing RNA from certain cell lines, but this will not affect the RNeasy procedure.

6 Apply up to 700 µl of the sample, including any precipitate that may have formed,

7 to an RNeasy mini column placed in a 2 ml collection tube (supplied). Close the tube gently, and centrifuge for 15 s at 14,000 rpm. Discard the flow-through. Reuse the collection tube in the next step. If the volume exceeds 700 µl, load aliquots successively onto the RNeasy column, and centrifuge as above. Discard the flow-through after each centrifugation step.

8 Add 350 µl Buffer RW1 to the RNeasy column. Close the tube gently, and centrifuge for 15 s at 14,000 rpm to wash the column. Discard the flow-through.

9 DNase treatment: Per sample add 10 µl DNase in 70 µl of Dnase-buffer and mix by gently inverting the tube. Pipet the DNase mix directly on the membrane of the column and let incubate for 25 minutes at room temperature. Any DNA that is present in the sample will now be digested.

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10 Wash the column by adding 700 µl of buffer RW1 to the column and centrifuge for 15 s at 14,000 rpm. Discard the flow-through and centrifuge another time for 15s at 14,000 rpm.

11 Transfer the RNeasy column into a new 2 ml collection tube (supplied). Pipet 500 µl Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 15 s at 14,000 rpm to wash the column. Discard the flow-through. Reuse the collection tube in step 9.

12 Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.

13 Add another 500 µl Buffer RPE to the RNeasy column. Close the tube gently, and centrifuge for 2 min at 14,000 rpm to dry the RNeasy silica-gel membrane. Transfer the RNeasy column into a new 2 ml collection tube and centrifuge for 1 minute at 14,000 rpm to eliminate any chance of possible Buffer RPE carryover. It is important to dry the RNeasy silica-gel membrane since residual ethanol may interfere with downstream reactions. This centrifugation ensures that no ethanol is carried over during elution.

14 To elute, transfer the RNeasy column to a new 1.5 ml collection tube (supplied). Pipet 30-50 µl RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube gently, and centrifuge for 1 min at 14,000 rpm to elute.

A.3.3. Determination of the integrity and size distribution of the RNA

The integrity and size distribution of the isolated RNA can be determined using denatured agarose gel electrophoresis and ethidium bromide staining. The ribosomal bands of the RNA have to appear as sharp bands on the gel. The intensity of the 28S (upper) band should be twice as high as the intensity of the 18S (lower) band. When this is not the case, or when the bands are not sharp, but appear as a smear or smaller sizes RNA, the RNA sample is denatured during the preparation.

Solutions

0.5 × TBE:

50 mL × 10 TBE 950 mL miliQ (not autoclaved)

10 × TBE:

108 g Tris 55 g boric acid 40 mL 0.5 M EDTA, pH = 8.0 Complete upto 1 L with double autoclaved miliQ

0.5 M EDTA, pH = 8:

37.2 g EDTA 200 mL miliQ First adjust the pH (with NaOH) before dissolving the solid. EDTA is an acid that only dissolves at high pH.

10× Ficoll Orange: 40 mg Orange G/10 ml

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2.5 g Ficoll 400/10 ml Store aliquots at -20 ˚C, working solution in the fridge.

DNA ladder, concentration 0,07 µg/µl:

100 µl stock 285,7 µl 10× Ficoll Orange 1042.9 µl ddH2O. Store aliquots at -20 ˚C.

Procedure:

Be aware that ethidium bromide is very carcinogenic! So directly after adding the chemical and pouring the gel, get rid of everything what could be in contact with it, like gloves, pipettips, tissues etc.. Do not touch anything before you have done that and do not walking around with the same gloves where you add the EtBr with!

− Prepare agarosegel: For 1% agarose gel, add 1 gram agarose to 100 ml 0.5× TBE buffer. Dissolve by heating the solution in a microwave. Cool down the solution until it is hand warm and add 5 µL ethidium bromide. Poor the solution in the mould, and place the comb. Let the solution polymerize for about 10-15 minutes.

− Fill electrophoresis system with 0.5× TBE. Place gel tray with agarose gel in the system (comb on negative/black site), remove comb and be aware the gel is covered by the buffer. You can re-use the buffer in the reservoir 5 times

− Load the agarose gel with the samples and DNA ladder. Mix 7 µl sample with 3 µl 10× ficoll orange by using 15-wells-comb and mix 5 µl sample with 2 µl 10× ficoll orange by using 20-wells-comb.Run the gel for about 15 minutes at 100-120 Volt. Note: DNA is charged negatively, and runs towards the plus (red) pole.

− The band can be visualized with UV-light. Save and/or print an image with software connected to the Biorad Versadoc system.

A.3.4. Quantification and purity of the RNA

− Start the program ‘Nanodrop’ on the computer

− Choose nucleic acid for DNA/ RNA assay

− Clean the pedestals with demi- water and tissue

− Load 1.5 µl water, and close the nanodrop carefully

− Click OK � start of initialization

− Choose sample type (DNA 40) at the upper right

− First, measure a blank: load 1.5 µl of a blank sample and click blank

− Clean the pedestals with demi- water and tissue

− Load 1.5 µl of your sample and click on measure

− Wipe your sample of and do the next measurement

− Click on exit when you are finished

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A.4. cDNA synthesis

Procedure:

1 Prepare Buffer/Primer mix according to the number of samples + blanc + 1 extra and mix well. Per reaction:

- 2.5 µl Random Primer - 2.5 µl dNTP’s 5mM - 5.0 µl 5×First-Strand Buffer

Total 10 µl mix per reaction Aliquot mix in 1,5 ml eppendorf tubes and add 11 µl (RNA + ddH2O) corresponding with 500 ng RNA or 11 µl only ddH2O for the blanc.

2 Total program:

a. 72 ˚C, 6 min b. 37 ˚C, 5 min Then add 4 µl of Enzyme Mix (see step 3) c. 37 ˚C, 1 hr d. 95 ˚C, 5 min e. 4 ˚C

3 During step a. and b. the Enzyme Mix is prepared according to the number of samples + blanc + 1 extra. Per reaction:

- 1.0 µl ddH2O - 2.5 µl DTT - 0.5 µl M-MLV enzym

Total 4 µl mix After step 2b. add Enzyme Mix, then go further with the rest of the program.

Store cDNA samples at 4 ˚C

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A.5. Real- time PCR

Principle

Nucleic acid amplification and detection are among the most valuable techniques used in biological research today. Real-time detection of PCR products is made possible by including in the reaction a fluorescent chemistry that reports an increase in the amount of DNA with a proportional increase in fluorescent signal. The fluorescent chemistries employed in this protocol include the DNA-binding dye SYBRGreen.

Figure A.5: Binding of SYBRGreen to dsDNA

SYBRGreen can be used with different primer combinations, but is not specific for gene of interest, it binds to all dsDNA. Real-Time analysis of PCR enables truly quantitative analysis of starting template copy number with accuracy and high sensitivity over a wide dynamic range. Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence as amplification occurs, here the MyiQ Single Color Real Time PCR Detection System is build on the iCyclerrThermal Cycler. The MyiQ optical design supports the use of a single filter pair optimized for excitation and emission of green fluorescent dyes, resulting in excellent sensitivity for the detection of fluorophores such as FAM and SYBR Green I. In both systems a CCD detector captures a simultaneous image of all 96 wells. This results in a comprehensive data set illustrating the kinetic behavior of the data during each cycle. Simultaneous image collection insures that well-to-well data may reliably be compared. The iQ5 software reports data on the PCR in real time, allowing immediate feedback on reaction success.

Solutions

To have not the risk of cross-contaminations, each person should prepare his/ her own working solutions.

− Double autoclaved Ultrapure water (ddH2O) or nuclease-free water.

− Primers are delivered in dry form. According to the delivery form dissolve each primer to a stock concentration of 100 µM with ddH2O, store 40 µl aliquots at -20 ˚C. Before use in PCR prepare a working solution of 20 µM: dilute 5× by adding 160 µl of ddH2O and store at 4 _C. This working solution can be used a several times, but prepare new after a couple of months.

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− The iQ SYBR Green Supermix is a 2× mix for real-time PCR applications. It is a readymade mixture of the hot-start enzyme iTaq DNA Polymerase, dNTP’s, buffer, SYBR Green I, fluorescein.

Procedure

1 Switch on: a. Biorad Thermal Cycler b. MyiQ Single Color Real Time Detection Upgrade c. computer iQsoftware

2 Generate with the iQsoftware in Workshop: Protocol and Plate (see for more details Help� Quick Guide or the paper manual) Protocol:

Cycle 1: (1×) 95 ˚C, 3 min

Cycle 2: (40×) 95 ˚C, 20 s

60 ˚C, 20 s 72 ˚C, 30 s Cycle 3: (1×)

95 ˚C, 1 min Cycle 4: (1×)

65 ˚C, 1 min Cycle 5: (61×)

65 ˚C � 95 ˚C, ∆T=0.5 ˚C, 10s, to obtain a melting curve Cycle 6: (1×) 4˚C, 10 min

3 Calculate and prepare the SybrGreenI/Primermix according to the number of

samples + blanc + 1 extra (if more than 15 reactions 2 extra, if more than 30 reactions 3 extra, etc.). Mix per reaction:

7.50 µl iQ SYBR Green Supermix 6.50 µl ddH2O 0.375 µl forward primer 20 µM 0.375 µl reverse primer 20 µM 1.00 µl cDNA

Use PCR tubestrips (less samples) or a 96-wells PCR plate (large amount of samples).

4 Put the prepared samples into the Thermal Cycler 5 Start the PCR in Software � Workshop � Run. Before it starts running fill in

(if necessary) some notes. Press oke, the run will start and data will be saved.