Riesgo de liquidez -

Real-time PCR is a technique used to detect and quantify the initial amount of the template. It monitors the fluorescence emitted by a reporter during each reaction cycle which its intensity is proportional to the amount of PCR product generated. Real-time PCR quantifies the abundance of transcript. The point at which the target sequence is detected is called the cycle threshold (Ct). The Ct value of a gene of interest is subtracted from the Ct of a housekeeping gene resulting in the ∆Ct value.

The oligonucleotide primers are complementary to the 5’ and 3’ ends of a region of interest amplifying cDNA. The presence of the fluorogenic probe which is chemically synthesised with a fluorescent reporter dye at the 5’ end and a quencher dye at the 3’ end allows quantification of the target transcript by fluorescence. The probe

72

binds to the DNA template and when the primer is extended, the probe is cleaved by the 5’-3’ exonuclease activity of Taq DNA polymerase. It leads to separation of the reporter dye from the quencher dye increasing the fluorescence emitted by the latter. Then, the probe is removed from the DNA template, and the primer extension continues until it reaches the end of DNA strand.

All real-time PCR experiments were carried out using Life Technologies gene expression assays (Life Technologies, Warrington, UK). The 18S rRNA was used as a housekeeping gene at a final concentration of 25 mM each. The control reactions were carried out in separate wells from target gene expression measurements (singleplex) in 96-well plates (Life Technologies, Warrington, UK). The reactions were set up in triplicate by combining the following components: 5 µL of 2x Master Mix, 0.5 µL of either 18S mixture or 20x expression assay, 100 ng of cDNA and nuclease free water to a final volume of 10 µL per well. Plates were sealed with a clear adhesive film (Life Technologies, Warrington, UK) and finally run on 7500 real-time PCR system (Life Technologies, Warrington, UK) for 40 cycles. All data was expressed as Ct values and used to calculate ∆Ct (Ct of gene of interest – Ct of 18S). They were expressed as arbitrary units (A.U. = 1000x2-∆Ct). Fold change was calculated using the 2-∆∆Ct method.

2.7 11β-HSD1 enzyme activity assay

This technique allows measurement of the interconversion between the rodent inactive 11-DHC (A) and active corticosterone (B) by 11β-HSD1. This protocol was carried out on both monolayers of intact cells, microsomes and whole tissue explants.

73

Confluent cell monolayers were washed once with PBS, then incubated in serum free medium containing 100 nM of either 11-DHC enriched with 20,000 cpm/reaction tritiated [1,2,6,7-3H(N)]-11-DHC (3H-11-DHC) (for in-house synthesis see section below) or 100 nM of corticosterone enriched with 20,000 cpm/reaction of tritiated [1,2,6,7-3H(N)]-corticosterone (3H-corticosterone) (GE Healthcare, Bucks, UK). Incubations were carried out at 37ºC under a 5% CO2 atmosphere for 30 mins (liver) and 2 h (quadriceps). Reaction medium was transferred to glass test tubes and 5 mL of dichloromethane was added. Steroids were extracted from medium by vortexing the tubes for 20 seconds followed by centrifugation at 1,000 g for 10 mins in order to separate aqueous from organic phases. Then the aqueous phase was aspirated, and the organic phase containing the steroids was evaporated at 55ºC using an air blowing sample concentrator (Techne, New Jersey, US). Steroids were re-suspended in 40 μL of dichloromethane, and using a Pasteur pipette they were spotted onto a silica coated thin layer chromatography plate (Thermofisher, Surrey, UK) followed by a non-radiolabelled 5 μL of 11-DHC/corticosterone (10 mM in ethanol). Each spot was separated by 1.5 cm from adjacent samples and 2 cm from the bottom of the plate. Thin layer chromatography (TLC) was used to separate the steroids in 200 mL of chloroform:absolute ethanol (92:8) mobile phase for 1.5 h. Radioactivity of the separated 3H-11-DHC/3H-corticosterone was measured using a Bioscan 200 imaging scanner (LabLogic, Sheffield, UK). To assign the radioactivity peaks with the correct steroids, the position of cold steroids, were visualised under UV light. The percentage conversion was calculated using region counts for the individual peaks and enzyme activity was expressed in pmoles of steroid converted per mg of protein per hour (pmol/mg/h).

74

Fresh tissue explants (~20 mg/well) or 100 µg muscle microsomes were added to 1 mL of serum free media and the above protocol was followed. Enzyme activity was expressed as pmoles of steroid converted per g of tissue per hour (pmol/g/h) or % conversion respectively.

2.7.1 3H -11-DHC production

Because tritiated 3H-11-DHC is not commercially available, consequently it was generated from 3H-corticosterone (GE Healthcare, Bucks, UK). 20 μL of 3H- corticosterone (1 mCi/mL) was incubated with 250 μg of homogenised mouse placenta in 500 μL of 0.1 M potassium phosphate buffer (80.2 mL of 1 M K2HPO4, 19.8 mL of 1 M KH2PO4, 900 mL of H2O), pH 7.4, with 500 μM NAD+

. Conversion was carried out at 37ºC in a shaking water bath overnight. Steroids were extracted by addition of 5 mL of dichloromethane. Tubes were vortexed for 20 s and centrifuged at 1,000 g for 10 mins to separate aqueous and organic phases. The aqueous phase was aspirated and the organic phase with steroids was evaporated at 55ºC using an air blowing sample concentrator (Techne, New Jersey, US). Steroids were re-suspended in 70 μL of dichloromethane, spotted onto a silica coated thin layer chromatography plate (Thermofisher, Surrey, UK) using a Pasteur pipette and separated by TLC using 200 mL of chloroform:absolute ethanol (92:8) as the mobile phase for 1.5 h. The position of 3H -11-DHC was established by reading the silica plates using a Bioscan 200 imaging scanner (LabLogic, Sheffield, UK). The silica at the 3H-11-DHC position was scraped into a glass tube and 300 μL of 100% ethanol was added for steroid elution. The tubes were incubated at 4ºC overnight. To separate the eluted 3H-11-DHC and silica, the tubes were centrifuged at 1,000 g for

75

5 mins. Quality and radioactivity of 3H-11-DHC were determined by separating 5 μL of stock by TLC. Number of counts was measured using the Bioscan 200 imaging scanner (Figure 2-3). Finally, the stock was diluted in ethanol to ~1,000 counts/μL which is equivalent of 1.5 pmol/µL.

Figure 2-3 Representative of Bioscan traces of 3H-11-DHC