Following primer design with Primer3 [21 (link)], the expression levels of 17 selected genes were defined by qPCR to validate the RNA-seq results and evaluate time-related expression trends (Table 2 ). The specificity of each primer pair was preliminary verified with BLAST searches.
Briefly, 1 μg of total RNA was retro-transcribed into cDNA using oligo(dT)12–18, 25 ng primers and 200 U of SuperScript II Reverse Transcriptase (Life Technologies). Quantitative PCR analysis was carried out with the Rotor Gene RG-3000A Real Time PCR system (Corbett Research, Sydney, Australia) using the SYBR Green I dye (Roche, Mannheim, Germany) and combined sense and antisense primers at 300 nM final concentration. We used the following cycling conditions: an initial denaturation step of 10 min at 95°C, 45 cycles of amplification consisting of 15 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C. cDNA samples were analyzed in triplicate. Gene expression was evaluated with ΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. GAPDH was identified also by sequencing data as the most stably expressed housekeeping gene in corneal samples [22 (link)]. Results are expressed as gene expression fold change of the treated corneas compared to the control ones.
Briefly, 1 μg of total RNA was retro-transcribed into cDNA using oligo(dT)12–18, 25 ng primers and 200 U of SuperScript II Reverse Transcriptase (Life Technologies). Quantitative PCR analysis was carried out with the Rotor Gene RG-3000A Real Time PCR system (Corbett Research, Sydney, Australia) using the SYBR Green I dye (Roche, Mannheim, Germany) and combined sense and antisense primers at 300 nM final concentration. We used the following cycling conditions: an initial denaturation step of 10 min at 95°C, 45 cycles of amplification consisting of 15 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C. cDNA samples were analyzed in triplicate. Gene expression was evaluated with ΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. GAPDH was identified also by sequencing data as the most stably expressed housekeeping gene in corneal samples [22 (link)]. Results are expressed as gene expression fold change of the treated corneas compared to the control ones.
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