Vic mgb

Cs shRNA and Con shRNA cells were homogenized in 1 ml of ice cold TRIZOL Reagent (Invitrogen Ltd, Paisley, UK) and RNA extracted using chloroform and isopropanol as described previously [6 (link)]. 2 μg of RNA was then used for cDNA synthesis in 20 μl reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 0.5 mM dNTP Mix (0.5 mM each dATP, dGTP, dCTP and dTTP), 5 mM DTT, 150 ng of random primers, and 200 units of SuperScript™ III Reverse Transcriptase. Real time PCR was performed using Roche Lightcycler 480 II (Roche Diagnostics, Sussex, UK) and Multiplex Taqman assays for Cs as a target gene and β-actin as a reference gene in each sample. Three μL of cDNA was added to 10 μL of LightCycler 480 Probe Master (Roche), 0.2 μL of TaqMan probe (Probe no. 100, Universal Probe Library), 0.2 μL of forward and reverse primers (20 μM) each, and 1 μL of mouse β-actin probe dye VIC-MGB (Applied Biosystems, 4326317E). The mouse Cs intron spanning primers were designed using Universal Probe library software and purchased from Sigma-Genosys (forward primer: 5′-GGAAGGCTAAGAACCCTTGG-3′ and reverse primer: 5′-TCATCTCCGTCATGCCATAGT-3′) and the corresponding UPL probe (UPL probe #100) was used. The results were analysed using LightCycler® 480 software 1.5 and Cs was normalized to β-actin and presented as a ratio (ratio = (1 + E_Cs)^−Ct(Cs)/(1 + E_β-actin)^{−Ct(β-actin)}).

Cell samples were homogenised in 1 ml of ice cold TRIZOL Reagent (Invitrogen Ltd, Paisley, UK) and RNA extracted using chloroform and isopropanol as described previously [8 (link)]. 2 μg of RNA was then used for cDNA synthesis in 20-μl reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 0.5 mM dNTP Mix (0.5 mM each dATP, dGTP, dCTP and dTTP), 5 mM DTT, 150 ng of Random primers and 200 units of SuperScript™ III Reverse Transcriptase. Real time PCR was performed using Roche Lightcycler 480 II (Roche Diagnostics, Sussex, UK) and Multiplex Taqman assays for Cs as a target gene and β-Actin as a reference gene in each sample. Three μL of cDNA was added to 10 μL of LightCycler^® 480 Probe Master (Roche), 0.2 μL of TaqMan^® probe (Probe no. 100, Universal Probe Library), 0.2 μL of forward and reverse primers (20 μM) each and 1 μL of mouse β-Actin probe dye VIC-MGB (Applied Biosystems, 4326317E). The mouse Cs intron spanning primers were designed using Universal Probe library software and purchased from Sigma-GenoSys (Forward primer: 5’-GGAAGGCTAAGAACCCTTGG-3’ and Reverse primer: 5’-TCATCTCCGTCATGCCATAGT-3’) and the corresponding UPL probe (UPL probe #100) were used. The results were analysed using LightCycler^® 480 software 1.5 and Cs was normalised to β-Actin and presented as a ratio (Ratio = (1 + E_Cs)^-Ct[Cs] / (1 + E_βactin)^{-Ct[β-actin]}) [18 (link)].