RNA isolation and qRT-PCR
Both injected and uninjected whole eyes were collected at PI-2, 7, 14, 21, 30, and 120 days for analysis of mRNA levels. qRT-PCR was performed with a MyIQ single-color qRT-PCR machine (Bio-Rad). Mice were euthanized, eyes were enucleated and homogenized and total RNA was extracted using TRIzol (Invitrogen Inc. Carlsbad, CA) as described previously [13]. Subsequently, DNase treatment was performed with RNase-free DNase (Promega Inc.) to remove both genomic DNA and any remaining nanoparticle DNA. cDNA synthesis by reverse transcription was performed and 20 ng of cDNA from each sample was used for qPCR. Rds primer sequences were reported previously [13]. Melting curve analysis and agarose gel electrophoresis were performed at the end of the reaction to ensure that the PCR products were specific and of appropriate size. All experimental mRNA levels were quantified against the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) as described previously [13]. Relative expression levels were calculated by the 2−ΔcT method [48]. At least three injected and three uninjected eyes from each treatment group at each of the scheduled time points were analyzed. Two independent qPCR experiments for each set of samples were performed and individual samples were run in triplicate. For each sample, the six values (three from two separate reactions) were averaged to get an expression value. The S.E.M. shown in Figure 1 represents the variation from sample to sample (i.e. inter-mouse variation). Rds primers amplify from transferred Rds and WT Rds, but not from the Rds mutant allele. To confirm that Rds levels were not artificially altered by the presence of undigested nanoparticle, control reactions amplifying from the IRBP or CBA promoter regions were performed and no product was detected. As an additional control, a subset of samples was analyzed with Rds primers, but without the addition of reverse transcriptase and no amplification was detected.