CONSIDERACIONES ESTÉTICAS SOBRE CÓRDOBA

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discarded. This step was repeated again but instead centrifuged for 2 minutes and afterwards the RNeasy Mini column was placed in a new 2mL collection tube and centrifuged at 16,000g for 1 minute. Finally to collect the isolated RNA the column was placed in a 1.5 mL

RNase-free tube and 30 µL of UltraPure™ DNase/RNase-Free dH2O (Gibco, 10977) added

to the column and centrifuged at 8,000g for 1 minute. RNA samples were stored at -80°C until required for further processing. All products were purchased from Qiagen (RNeasy Mini Kit) unless stated otherwise.

2.6.2.3 Reverse Transcription

A RT2 _{First Strand Kit (QiaGen, 330401) containing 5X gDNA Elimination Buffer (GE), 5X}

Reverse Transcription Buffer (BC3), RT Enzyme Mix 3 (RE3), Primer and External Control

Mix (P2), and RNase-free H2O was used to synthesise cDNA from isolated RNA. A

Genomic DNA Elimination Mixture was prepared by adding 8 µL RNA sample to 2 µL of GE buffer in a sterile PCR tube. Contents were mixed gently with a pipette followed by brief centrifugation and incubated at 42°C for 5 minutes. Next, tube was chilled on ice immediately after for one minute. Parallel, the RT cocktail was prepared by adding 4 µL

BC3 Buffer, 1 µL P2 Mix, 2 µL RE3 Mix, and 3 µL H2O to a sterile PCR tube. First strand

cDNA synthesis was executed by mixing 10 µL of RT cocktail gently with the 10 µL Genomic DNA Eliminiation Mixture, incubated at 42°C for exactly 15 minutes and immediately followed by incubation at 95°C for 5 minutes to terminate the reaction. The resulting cDNA was stored at -20°C until required for further processing.

2.6.2.4 RT-PCR

The synthesised cDNA was either diluted 10 or 100 fold with UltraPure™ DNase/RNase-

Free dH2O for subsequent RT-PCR reactions. Prior to RT-PCR reactions the following

solutions were prepared. Stock Primer Mixes were prepared from 100 µL Sense Primer and 100 µL of associated Anti-Sense Primer (Invitrogen) according to Table 2.3. Subsequently, a PCR Reagent Stock solution was created from 7.5 µL iQ SYBR Green Supermix (Bio-Rad,

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170-8882), 4.5 µL UltraPure™ DNase/RNase-Free dH2O, and 1 µL Stock Primer Mix per

well. All PCR reactions were carried out in iQ 96-Well PCR plates (Bio-Rad, 223-9441).

Diluted cDNA was added to each well in triplicates of 2 µL and 13 µL of PCR Reagent Stock solution was added per well with cDNA to obtain a total working volume of 15 µL. Well content was gently mixed using a pipette whilst avoiding air bubbles. Negative controls were included for each Primer Stock Solution by replacing the cDNA with UltraPure™

DNase/RNase-Free dH2O, in triplicate.

RT-PCR reactions were carried out using a Bio-Rad iCycler (Version 2.039) and the standard iCycler operating platform (Bio-Rad). The PCR profile included a 3 minute 95°C initialisation step followed by 40 cycles of a 2 two-step amplification programme: 30 seconds at 95°C (denaturation) and 30 seconds at the optimum annealing and extension

temperature for each specific primer (Table 2.2, Tm). Threshold cycle (Ct) values were

calculated when the fluorescence from SYBR Green binding to double stranded DNA entered its exponential phase during amplification. Subsequently, PCR products were subjected to melting curve analysis.

Target Gene Sense Primer- nanomoles Sense Primer- Volume (µL) Antisense Primer- nanomoles Antisense Primer- Volume ( µL) GAPDH 18.0 180 24.3 243 CD34 17.6 176 13.9 139 GATA2 23.5 235 18.6 186 HoxB4 11.8 118 13.0 130 Hb-ß 17.8 178 21.6 216 Tal1 21.4 214 21.2 212 RunX1 20.6 206 21.7 217 EPO 25.3 253 23.7 237

52 2.6.3 Histology

2.6.3.1 Preparation of a blood/cell smear

Cell suspensions were diluted to appropriate cell densities for analysis (i.e. whole blood was

diluted 1:2 in PBS). A drop of cell suspension (5-20µL) was placed near the edge of the glass slide. Another glass slide, the spreader, was used to create a smear by placing it at a 45° angle and backing into the drop of cell suspension so that the drops spread evenly along the edge before pushing the spreader across the slide to create the smear. The slide with smear was air dried before the staining procedure was started.

2.6.3.2 Brilliant cresyl blue stain for reticuloctyes

Brilliant cresyl blue (Fluka, Sigma-Aldrich) stock solution was diluted 1:80 to 1:100 with PBS. For a single test 20 µL blood or 20 µL cell suspension was added to 20 µL brilliant cresyl blue (BCB) solution in a tube. This solution was mixed thoroughly and a thin smear was prepared after 30 minutes of incubation at room temperature. For test in series, thin smears of BCB solution were prepared on microscope slides. Air-dried slides were stored up to 2-3 weeks. When used, a small drop of blood was quickly smeared over the stain layer and the still wet preparation was placed in a moist chamber for 5-10 minutes before air drying. Samples were observed using light microscopy (Zeiss).

2.6.3.3 Methylene blue stain for observation of cell nucleus

Glass slides with air dried cell smear were subjected to staining using a 0.05% solution of

methylene blue in dH2O (Sigma-Aldrich). Samples were incubated with the stain for 5

minutes at room temperature followed by rinsing in dH2O. Samples were air dried at room

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2.6.3.4 Preparation of cells for scanning electron microscopy (SEM)

Prior to cell seeding, cover slips (Sarstedt, 83.1840.002) were coated with Poly-L-Lysine (Sigma, P4832) for 5 minutes. The solution on the coverslip was discarded and cover slips

were washed with PBS and air dried for 2 hours. Cells (1x104_-1x106_{) were seeded on the}

coated cover slips. Next, cells were fixed with 1% (w/w) gluteraldehyde (Sigma, G5882) in PBS for 1 hour, cells were washed 3 times for 5 minutes with PBS followed by 1 wash in deionised water. Cells were then dehydrated using a graded ethanol series; 15%, 25%, 50%, 50%, 70%, 2x 100% (v/v) ethanol for 10 minutes each. Cover slips were dried with hexamethyldisilazane (Fluka, 52620) for 3 minutes prior to mounting onto aluminium specimen stubs (Agar Scientific Ltd) using carbon double sided sticky tabs (Agar Scientific Ltc). Afterwards, these specimens were gold sputter coated (Emitech K575X Sputter Coater) and analysed using a scanning electron microscope (LEO 1550).

2.6.4 Statistics

Statistical analyses were carried out using independent sample T-tests using the software package Microsoft Excel. All used p-values were stated with the corresponding results (Chapter 3 and 4). All error bars shown were calculated using the ‘STDEV’ formula (Standard Deviation) in the software package Microsoft Excel.

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Chapter 3