Optimal RNA and cDNA dilutions were determined by RT and subsequent PCRs on a seven-step, twofold dilution series of RNA (12.5, 25, 50, 100, 200, 400, and 800 ng RNA/RT) and inspecting for single cycle shifts in cycle threshold (Ct) between dilutions. Using more than 200 ng RNA per RT decreased 18S Ct differences, implying incomplete RT of 18S at high RNA amounts, and using less than 50 ng RNA per RT increased Ct for genes of interest. For each RNA sample, 100 ng total RNA was reverse transcribed (Quantitect Reverse Transcriptase; Qiagen) in a final 20-μL volume according to the manufacturer’s instructions. The resultant cDNA was diluted 20-fold, and 5 μL diluted cDNA (equivalent of 1.25 ng RNA/PCR) was used in a real-time PCR reaction with 100 nM (
Fgf1 and
Gdnf) or 200 nM (
18S,
Bdnf,
Cntf,
Fgf2,
Igf1) each of a primer pair (
18S forward, 5′-GTTGGTTTTCGGAACTGAGGC-3′,
18S reverse, 5′-GTCGGCATCGTTTATGGTCG-3′;
Bdnf forward, 5′- AACCAGAAAAAGCACCAAAA-3′,
Bdnf reverse, 5′- CTTGTGCTGAATGGACAGAA-3′;
Cntf forward, 5′-GGACCTCTGTAGCCGTTCTA-3′,
Cntf reverse, 5′-TCATCTCACTCCAACGATCA-3′;
Fgf1 forward, 5′-AACCCAAACTGCTCTACTGC-3′,
Fgf1 reverse, 5′- GAGCCGTATAAAAGCCCTTC-3′;
Fgf2 forward, 5′-GCGGCTCTACTGCAAGA-3′,
Fgf2 reverse, 5′-CGTCCATCTTCCTTCATAGC-3′;
Gdnf forward, 5′-CCATGTTCCTAGCCACTCTG-3′,
Gdnf reverse, 5′- AGGCTGAAGTTGGTTTCCTT-3′;
Igf1 forward, 5′-TGGACGCTCTTCAGTTCGTG-3′,
Igf1 reverse, 5′-GTTTCCTGCACTTCCTCTAC-3′), 12.5 μL of 2× reaction cocktail (iQ SYBR Green Supermix; Bio-Rad, Hercules, CA) and nuclease-free dH
2O to 25 μL. Primers were designed with Primer3 (http://fokker.wi.mit.edu/primer3/).
41 cDNA was amplified in 96-well plates on a thermal cycler (MyIQ; Bio-Rad). Conditions were 95°C, 3 minutes; 40 cycles of 94°C, 30 seconds denaturation; 55°C, 30 seconds annealing; 72°C, 30 seconds extension; 72°C, 7 minutes, with fluorescence recorded at the end of each 72°C extension. Melt analysis conditions were heating from 55°C to 95°C in 0.5°C increments, 15 seconds per increment, with fluorescence recorded at each increment. The Ct of each reaction was determined from a PCR baseline-subtracted and curve-fit option (iCycler; Bio-Rad). Three real-time PCRs were performed for each cDNA sample-primer pair combination, and the average Ct was calculated. Taking 18S as the endogenous control, growth factor transcript abundance (expression) relative to control eyes or the opposite eye was calculated from the average PCR cycle thresholds using the 2
−ΔΔCt method of Livak and Schmittgen.
42 The expression ratio was computed to minimize animal-to-animal variability in gene expression.