Rotor gene rg 3000a real time pcr system

Total RNA from tissue and cells were extracted using RNAiso Plus (Takara) and reverse transcribed into cDNA with a QuantiTect Reverse Transcription Kit (Qiagen). Quantitative RT‐PCR was done with Rotor‐Gene SYBR Green Kit with a Corbett Rotor‐Gene RG‐3000A Real‐Time PCR System. mRNA was quantified using the comparative threshold cycle method with beta‐actin as control. Primers used were listed in the Table S2 and Table S3.

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.

Total RNA was extracted using RNAiso Plus according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). After an initial amplification with a denaturation step at 95°C for 5 m, followed by 30–40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 10 s, and extension at 72°C for 30 s, a final extension at 72°C for 5 m was done upon completion of the cycling steps. The cDNA was amplified in duplicate using a Rotor-Gene SYBR Green RT-PCR Kit (Qiagen) with a Corbett Rotor-Gene RG-3000A Real-Time PCR System. The data were evaluated using the RG-3000A software program (version Rotor-Gene 6.1.93, Corbett, Sydney, Australia). The sequences of primer pairs were described as follows: IL-1β: 5′-CAACCAACAAGTGATATTCTCCATG-3′ and 5′-GATCCACACTCTCAGCTGCA-3′; inducible nitric oxide synthase (iNOS): 5′-GCCACCAACAATGGCAAC-3′ and 5′- CGTACCGGATGAGCTGTGAATT- 3′; TNF-α: 5′-ATGGCCTCCCTC TCAGTTC -3′ and 5′-TTGGTGGTTTGCTACGACGTG-3′; and IL-10: 5′-GACCAGCTGGACAACATACTGC TAA-3′ and 5′-GATAAGGATTGGCAACCCAAGTAA-3′. For data normalization, an endogenous control (actin) was assessed to control for the cDNA input, and the relative units were calculated by a comparative Ct method. All qRT-PCR experiments were repeated three times, and the results are presented as the means of the ratios ± SEM.