Ready to go you prime first strand beads

Corneal epithelium and conjunctiva were extracted to measure gene expression of inflammatory mediators via PCR. Corneal epithelium was scraped with a scalpel and conjunctiva was surgically excised. Tissue samples from cornea and conjunctiva were pooled separately from both eyes to give one cornea and one conjunctiva sample per mouse. RNA was extracted using the RNeasy Plus Micro kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Concentration of isolated RNA was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). After RNA isolation, cDNA was synthesized using Ready-To-Go You-Prime First-Strand beads (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) as previously reported.^{17 (link)}

Using a RNeasy Plus mini kit (QIAGEN, Valencia, CA, USA), total RNA was extracted from tumor samples that had been frozen and stocked within 30 mins after resection. Total RNA was reverse transcribed to cDNA using a Ready-To-Go You-Prime First-Strand Beads (GE Healthcare Life Sciences, Pittsburgh, PA, USA). For PCR amplification, each cDNA was diluted to 10 ng/μL. PCR conditions were as follows: p53 exon 4 forward: 5ʹ-CCC AAG CAA TGG ATG ATT TG-3ʹ; p53 exon 10 reverse: 5ʹ-AGC CTG GGC ATC CTT GAG-3ʹ. The PCR assay was carried out in a 15μL volume that contained 15 ng of cDNA and 1 unit of Taq PCR Master Mix Kit (QIAGEN). Each PCR reaction was started at 95°C for 5 mins, and then cDNA was amplified for 40 cycles at 95°C for 30 s, 54.7°C for 30 s, and 72°C for 90 s, with a final extension time of 7 mins at 72°C. Each amplicon was purified using a QIAquick Gel Extraction Kit (QIAGEN) after agarose gel electrophoresis. Purified PCR products were sequenced in forward and reverse directions using a 3130xl Genetic Analyzer (Thermo Fisher Scientific K.K.). We detected p53 mutations in exons 5 through 8, similar to previous reports.25 (link),26 (link)