This section, and 2.7.2 describe the methods used in Chapter 4 human in vitro experiments.
HCASMC were seeded at a density of 5x104 cells per well in a 12-well plate using normal SMC media (see section 2.3) and incubated overnight at 37oC to achieve ~80% confluency. The media was removed and cells washed once with PBS. To each well, 1ml of serum-free (0.1%) high-glucose DMEM was added and the plates incubated for a further 48h (quiescence) to induce a differentiated contractile state.
The cells were then stimulated to a dedifferentiated proliferative phenotype by replacing media with the following mitogens (in duplicate):
• No serum (serum-free DMEM) as an undifferentiated control • 15% serum (in normal SMC media)
• PDGF-BB at 20ng/ml (stocks at 10µg/ml so 2µl added to 1ml media)
• IL-1α at 10ng/ml (stocks at 1µg/ml [made up in 0.1% BSA/PBS as per manufacturers instructions] so 10µl stock added to 1ml media)
• bFGF at 20ng/ml (stocks at 10µg/ml so 2µl added to 1ml media)
Each plate was then incubated at 37oC for a variable duration of time dependant on the time-point being studied (1h, 5h, 18h or 24h). At the appropriate time point, cells were washed again with PBS and 700µl QIAzol (QIAGEN) added to allow cell harvesting and extraction of RNA.
2.7.2 Cell migration
HCASMC were seeded in 12-well plates and quiesced in 0.1% media for 48 hours as described in section 2.7.1. A previously described scratch assay was then used as this has been shown to effectively and simply mimic cell migration in-vivo (Liang et al., 2007). This method is based on the observation that, upon creation of a new artificial gap (a scratch or wound) on a confluent cell monolayer, the cells on the edges of the newly created gap will move together to close the scratch until new cell–cell contacts are established again. In this thesis, the purpose of this assay was not to directly measure the degree of migration obtained, but to promote a migratory cell phenotype and measure associated changes in microRNA expression. Direct quantification of the degree of migration was therefore not obtained and microscopy was only used to confirm the presence of cell migration at each time-point. A 200µl pipette tip was used to produce three evenly sized vertical scratches per well. Cells were then washed with PBS, placed in fresh media and returned to the incubator. Images were captured at 0, 6 and 16 hours post- scratch. At the appropriate time point, cells were washed again in PBS and harvested by the addition of 700µl QIAzol (QIAGEN) as in section 2.7.1.
2.7.3 RNA extraction
The following sections describe methods used in Chapter 4 for the quantification of miR both in vitro and in vivo.
2.7.3.1 Total RNA extraction from cells
RNaseZap (Ambion, TX, USA), a surface decontamination solution that destroys RNases on contact, was used to clean all apparatus and work surfaces. RNase-free filter tips were used in all experiments using RNA. Total RNA (including miRNA and small RNA fractions) was extracted from cells using the miRNeasy Mini Kit (QIAGEN) as per
manufacturer’s instructions. Briefly, cells were harvested in QIAzol, transferred to RNase- free 1.5ml eppendorfs and 140µl of cholorform was added. The samples were mixed by 15s of vigorous shaking. Following a 2-3 min incubation at room temperature, the homogenate was centrifuged at 12,000 x g for 15 min at 4oC. The upper aqueous phase was added to a new collection tube (care taken to avoid transfer of any interphase) and 1.5 volumes (usually ~525µl) of 100% ethanol added to ensure ideal binding conditions. The samples were then mixed thoroughly by pipetting and up to 700µl transferred into a RNeasy Mini filter column in a 2ml collection tube. After centrifugation at > 8000 x g for 15 secs at room temperature the flow-through is discarded. This centrifugation allows the RNA to bind to the silica-gel membrane of the spin column. This spin is repeated until all of the sample (in aliquots of up to 700µl) has passed through the membrane. At this point, on-column DNAse treatment was performed using the RNase-Free DNase Set (QIAGEN) as per the manufacturer’s instruction. Briefly, 350µ L of buffer RWT was added to the column and centrifuged for 15 secs at 8000 x g to wash. The flow-through was again discarded. 10µ L of DNAse I stock solution was added to 70µL of buffer RDD in a clean eppendorf and mixed by gentle inversion. This DNase I incubation mix was then pipetted directly onto the RNeasy Mini spin column membrane and incubated on ice for 15 min. A further wash step, adding 350µ L of buffer RWT into the column and centrifuging for 15s at 8000 x g was then performed and the flow-through discarded. Returning to the protocol, two further wash steps using 500µ L of buffer RPE with spins of 8000 x g for 15s and 2 min respectively are followed by elution of the RNA into 30µl of RNase-free water in a fresh collection tube (1.5ml eppendorf). The eluted RNA was passed through the spin column again to increase the RNA yield. The quantity of total RNA in each sample was quantified by NanoDropTM (ND-1000 spectrophotometer [Labtech International, Ringmer, UK).
2.7.3.2 Total RNA extraction from tissue
RNA extraction from porcine tissue was perfomed using the above method with some modifications. Frozen vessels were disrupted under liquid nitrogen in a pestle and mortar. Any stent present was removed at this point using careful dissection. The arterial fragments were homogenized (Polytron, Switzerland) in a bijou containing 1400µl of QIAzol (Qiagen) with intermittent cooling of the sample on ice. The homogeniser blade had been cleaned with 3% H2O2 for 30 mins before and after use and then washed with Nuclease
free water prior to use. Once completely homogenized, the sample was left to incubate for 5 min at room temperature before the addition of 280µl of cholorform and thorough
mixing. From this point on the method followed was as documented above for extraction from cells.
2.7.4 cDNA synthesis
For miRNA expression analysis, cDNA was synthesized from total RNA using stem-loop reverse transcription primers and RT kit (Applied Biosystems, Foster City, CA, USA). Each reaction utilises an RT primer specific to an individual miR. The RT product therefore contains single-stranded cDNA complimentary to the miR of interest only. Total RNA was diluted into aliquots at 2ng/µl concentration using RNase-free water. RT was performed in a 96-well PCR plate (Thermo Scientific) and is prepared on ice. Each well contains 2.5µl of total RNA (5ng) in addition to the following: 0.075µl of 100mM dNTP mix (with dTTP), 0.5µl MultiScribeTM Reverse Transcriptase (50U/µl), 0.75µl 10x RT buffer, 0.1µl RNase Inhibitor (20U/µl), 2.08µl Nuclease-free water and 1.5µl of specific miR (or endogenous control) primer. The plate was then mixed by gentle centrifugation and run on a PCR block. Thermal cycling conditions were 16oC for 30 min (to allow for primer annealing), 42oC for 30 min (to allow for cDNA synthesis) then finally 85oC for 5 min to stop the cDNA synthesis reaction and to destroy any DNase activity. Plates were then stored at -20oC until required for qRT-PCR.
2.7.5 Quantitative Real Time Polymerase Chain Reaction
TaqManTM qRT-PCR (Applied Biosystems, ABI Prism, 7900HT Sequence Detection System) was used to quantify the relative concentration of miRNA present in all experiments (cells and tissues) in this thesis. This quantitative process is based on the detection of a fluorescent signal produced proportionately during amplification of a PCR product. During the target amplification step, the AmpliTaq Gold DNA polymerase amplifies target cDNA synthesized from the RNA sample (section 2.7.4) using sequence- specific primers. The subsequent cleavage of TaqMan probes releases fluorescent signals. The amount of fluorescence released during the amplification cycle is proportional to the amount of product generated in each cycle and can be measured directly. When PCR amplification is in the exponential phase then data is acquired. qRT-PCR was performed using TaqMan Universal Master Mix II with TaqMan miR/endogenous control expression probes (Applied Biosystems) according to the manufacurer’s instructions. Reactions were performed using 384-well PCR plates and optical lids. Each well contained 5µl TaqMan Master Mix, 0.5µl miR-specific probe, 3.835µl nuclease-free water and 0.67µl of RT
product (section 2.7.4). Reactions were performed in triplicate and plates were set up on ice. 3 extra wells for each probe were added to the plate (with 0.67µl nuclease-free water instead of cDNA) as negative template control. Expression was normalized to miR-199a- 3p or RNU-48 for pig and human samples respectively. The plates were run using the TaqMan detection system under the following cycle conditions: 40 cycles of 95oC for 15s (amplification) then 60oC for 1 min (annealing). Delta(d)-Ct values were determined by subtracting the Ct of the appropriate endogenous control from the Ct of the miR in question. A reference sample is determined and the ddCt calculated by subtracting the reference value from each sample. The RQ is then determined by raising ddCT to power of -2 and raising negative ddCT to power of 2 (Schmittgen and Livak, 2008).