Vitreous samples were investigated for their levels of Fas, FasL, and TRAIL mRNA. Because of limited sample size, only 21 PVR, 9 RD, and 10 MH vitreous samples were investigated for TRAIL. Primers were designed using the program Primer 3 (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/; provided in the public domain by the Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA). All primers were cross-checked against the GenBank database to ensure no cross reactivity with other known human sequences (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). Primer pairs, one of which was fluorescent-dye–labeled, were synthesized by MWG Biotech (Ebersberg, Germany). All primer pairs were validated by sequencing of PCR products generated under the conditions stated. Primer sequences are detailed in
1 .
To correct for variation in amplification efficiency between individual reactions, target cDNA was coamplified with an internal competitor (mimic) of known concentration, using the same fluorescence-labeled primers. These mimics were prepared by PCR mutagenesis to delete 5 bases of sequence, 5 bp 3′ of the primer binding site, resulting in a PCR product 5 bases shorter than that of the original template cDNA. The mimic PCR products were cloned into pT7 blue (Novagen, Nottingham, UK) and verified by sequencing.
PCR amplification was performed by adding 1 μL of each cDNA sample to a final reaction mixture of 25 μL containing 60 mM Tris-Cl (pH 8.0), 15 mM (NH4)2SO4, 2 mM MgCl2, 0.2 mM each dNTP, 0.01% Tween 20, 0.5 U Taq polymerase (AmpliTaq Gold; Perkin Elmer, Warrington, UK), and 0.2 μM each primer; 103 single strands per reaction of appropriate mimic was added. Amplification cycles (performed on a Progene instrument; Techne, Cambridge, UK) were 94°C for 10 minutes, then 37 cycles of 94°C for 1 minute, 54°C for 1 minute, 72°C for 1 minute 30 seconds followed by 72°C for 15 minutes. Hypoxanthine phosphoribosyl transferase (HPRT), a constitutively expressed housekeeping gene was used to normalize the amount of mRNA present in each sample. Fluorescent-labeled PCR products were separated and analyzed by capillary electrophoresis under denaturing conditions (POP4 polymer) on a gene analyzer (Prism model 310; Applied Biosystems [ABI], Foster City, CA). Run conditions were a 5-second injection at 15 kV, run 24 minutes at 15 kV, at 60°C, on a 36-cm capillary (length to detection). Size and area of DNA peaks were obtained using standard software (Genescan version 2.0 and Genotyper v1.1.1; ABI). The amount of each cytokine mRNA in the samples was calibrated by the known concentration of mimic, using the following formula: Unknown concentration = (area of unknown/area of mimic) × mimic concentration.