Real-time PCR primers to detect rat fibronectin, laminin-β1, and PDGF-BB were designed using computer software (GCG Software Prime; Accelrys, Campbell, CA). Primers were chosen to generate an amplicon smaller than 150 bp. The sequences of the PCR primer pairs (5′ to 3′) that were used for each gene are as follows: rat fibronectin, 5′-GGGATCAAAGGGAAACACAG-3′ (forward) and 5′-AGACGGCAAAAGAAAGCAG-3′ (reverse); laminin-β1, 5′-TGTAGATGGCAAGGTCTTATTTCA-3′ (forward) and 5′-CTCAGGCAGTTCTGTTTGATGT-3′ (reverse); and PDGF-BB, 5′-GGGAATACTGCTCACAACG-3′ (forward) and 5′-GCATACAAAATAGCACTTCCG-3′ (reverse). Real-time PCR reactions were then performed with a PCR mix (iQ SYBR Green Supermix, containing 100 mM KCl, 40 mM Tris-HCl [pH 8.4], 0.4 mM of each dNTP, 50 U/mL DNA polymerase [iTaq], and 6 mM MgCl2, SYBR Green I, 20 nM fluorescein, and stabilizers; Bio-Rad, Hercules, CA). Thermocycling was performed in a final volume of 25 μL (8 μL DEPC H2O, 2 μL cDNA, 1.25 μL = 500 nM of each primer, and 12.5 μL of 2× iQ SYBR Green Supermix; Bio-Rad) using the PCR conditions of initial heating at 95°C for 300 seconds to denature cDNA and activate the Taq DNA polymerase, followed by 45 cycles consisting of denaturation at 95°C for 20 seconds, annealing at 58°C for 20 seconds, and extension at 72°C for 20 seconds using thermocycler (Smart Cycler; Cepheid, Sunnyvale, CA). One additional step, a melting curve, was added to determine specificity. The melting curve was constructed by increasing the temperature from 60°C to 95°C with a temperature transition rate of 0.2°C/s. To ensure that the correct product was amplified, all samples were separated by 1.2% agarose gel electrophoresis.
To correct for differences in both RNA quality and quantity between samples, data were normalized by using the ratio of the target cDNA concentration to that of GAPDH. To calculate the increases (
x-fold) in steady state RNA levels, the change in cycle threshold Δ
C T for control rat retina expression of fibronectin and laminin-β1 was calculated by subtracting from their threshold (
C T) the corresponding GAPDH Δ
C T (internal control). Then Δ
C T was calculated by subtracting the average of the Δ
C T in the control rat retina from the Δ
C T in sympathectomized rat retinal expression of the gene of interest. Changes in steady state gene expression are reported as
x-fold increases (2
−ΔΔCT) relative to control rat retina. The statistical analysis of steady state RNA levels is based on previous work by other groups.
12 13 Significance in the 2
−ΔΔCT was set at
P < 0.05, determined by computer (Prism Software; GraphPad, San Diego, CA).