Molecular biological methods

Small scale preparation of DNA was performed for quality control of the lentiviral plasmids containing the respective shRNAs to knockdown ROCK1 and both kinases.

This was performed using the Exprep Plasmid SV mini kit (GeneAll) following recommended manufacturer instructions.

Large scale preparation of DNA was performed to isolate the lentiviral plasmids containing the respective shRNAs to knockdown ROCK1 and ROCK2 for transfection into Tsa201 cells. For this, XChange Plasmid Maxi Kit (Peqlab) was used and protocol followed according to the manufacturer’s instructions.

The concentration and purity of isolated nucleic acids were measured using spectrophotometric quantification with the NanodropTM ND-1000 system. DNA concentration was calculated as follows:

A260: Absorbance at 260 nm A260 of 1.0 = 50µg/ml pure dsDNA

To evaluate DNA purity, the ratio of the absorbance at 260 nm divided by the reading at 280 nm was calculated. Good quality DNA will have an A260/A280 ratio of 1.7–2.0.

Samples were stored at -20°C until required.

3.4.2 Restriction digest

For quality control of lentiviral plasmids, plasmid DNA was digested using BamHI and NdeI (NEB). 10 U of the respective restriction enzyme was added to 1x reaction buffer and 1 μg of vector DNA. Water was added to a final volume of 30 μL and the reaction mix was incubated for 4 h, or overnight at 37°C. Restriction enzymes were inactivated at 65°C for 20 min.

3.4.3 DNA agarose gel electrophoresis

DNA agarose gel electrophoresis was used to separate and analyse DNA fragments based on their size and charge. To prepare a 1% agarose gel, 1 g agarose was mixed with 100 mL of 1x TAE buffer. After heating the mixture to melt the agarose and cooling it down to 65°C, 0.5 µg/µL of ethidium bromide was added and the solution was poured onto the gel support. DNA samples were separated in 1x TAE buffer at 120 V. DNA bands were visualized at 312 nm in the GelDoc XR imaging system (Biorad). Fragment size was estimated using DNA standard markers (see 2.4).

3.4.4 RNA isolation from cells and engineered tissues

Isolation of total RNA from cultured cells was performed using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions.

For isolation of RNA from engineered tissues, the phenol-chloroform method was used. For this, 1 mL TriFast(peqGOLD) was added per tissue and incubated at 4°C overnight to dissolve the collagen hydrogel. The following day, remaining pieces of tissue were mechanically dissociated by vigorous pipetting and subsequent RNA isolation as per the manufacturer’s protocol. To further clean up the RNA, the clean-up protocol of the RNeasy Mini Kit was used.

Isolated RNA was eluted in the appropriate amount of RNase-free H2O.

Concentration and purity of isolated RNA was measured using spectrophotometric quantification by the NanodropTM ND-1000 system. RNA concentration was calculated as follows:

A260: Absorbance at 260 nm A260 of 1.0 = 40µg/ml pure ssRNA

RNA had an A260/A280 ratio between of 1.9–2.1. Samples were stored at -80°C until required.

3.4.5 Formaldehyde agarose gel electrophoresis

Formaldehyde agarose gel electrophoresis was performed to verify the integrity of RNA samples. To prepare a 1.2% formaldehyde gel, 1.2 g agarose were mixed with 10 mL of 10x FA buffer and 100 mL of RNase-free H2O. After heating the mixture to melt the agarose and cooling down to 65°C, 1.8 mL of 37% formaldehyde and 0.5 µg/µL Ethidium bromide were added. The solution was then poured onto the gel support.

Prior to performing the electrophoresis, the gel was equilibrated in 1x FA gel running buffer for at least 30 min. For RNA sample preparation, 1 volume of 5x RNA loading buffer was mixed to 4 volumes of RNA sample. Before sample loading into the equilibrated gel, samples were incubated for 3-5 min at 65°C and then chilled on ice.

RNA samples were separated in 1x FA gel running buffer at 120 V for at least 45 min. RNA bands were visualized at 312 nm with the GelDoc XR imaging system (Biorad).

3.4.6 cDNA synthesis

Isolated RNA (see 3.4.4) was used to transcribe RNA into copyDNA (cDNA) using the RevertAid™ First Strand cDNA Synthesis kit (Fermentas) according to the manufacturer’s instructions. All steps were performed on ice. The composition of the cDNA master mix and the reverse transcription protocol are outlined in Table 28. The cDNA samples were stored at -80°C.

Table 28: Composition of cDNA master mix and reverse transcription protocol

Master mix components Amount

Template RNA 150 ng

Oligo (dT)18 primer 1 µL

 Incubate at 65°C for 5 min

5x reaction buffer 4 µL

RiboLock RNase inhibitor (20 U/µL) 1 µL

10 mM dNTP mix 2 µL

RevertAid M-MuLV RT (200 U/µL) 1 µL

 incubate at 42°C for 60 min, terminate reaction at 70°C for 5 min

3.4.7 Quantitative real-time polymerase chain reaction (qRT-PCR)

Quantitative real-time PCR was used to simultaneously amplify and semi-quantify cDNA. Non-specific fluorescent dyes, such as the EvaGreen^® dye (Solis Biodyne) The fluorescence measured after each elongation cycle is thus proportional to the

amount of cDNA in the reaction. The normalized target gene expression level was calculated as follows using the comparative Ct method (∆∆ct method):

All measurements were performed in quadruplicates. The final reaction was mixed as shown in Table 29. The PCR program is shown in Table 30. The list of the qRT-PCR primer sets can be found in 2.11.

Table 29: Master mix for qRT-PCR per 1 µL cDNA

Reagent Volume

cDNA (1:40) 1 µL

Primer for (10 pmol/µL) 1 µL

Primer rev (10 pmol/µL) 1 µL

Template DNA /Standard DNA (1:20) 1 µL

RNase-free water 13 µL

5x EvaGreen^® dye 4 µL

Table 30: qRT-PCR cycling program

Cycle Temperature Time Number of Cycles

Initial denaturation 95°C 15 min 1

Denaturation 95°C 15 s

40

Annealing 60°C 20 s

Elongation 72°C 40 s

Dissociation curve 95°C 15 s

60°C 15 s 1

95°C 15 s