Analysis of real-time PCR arrays data

To identify the most stable reference genes across the study group, RefFinder [53], a web-based comprehensive tool (http://fulxie.0fees.us) was used to rank the analyzed reference genes: GAPDH, RPLP0, SF3A1, B2M, and TBP. The GAPDH, RPLP0, and SF3A1 were identified as the most stable reference genes and used for normalization.

RT² Profiler PCR Array Data analysis web-based software v. 3.5 (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php), which is dedicated to the data analysis of PCR arrays, was used. Any Cq value >35 was considered undetected. The ΔCq for the gene of interest was calculated by subtracting the geometric mean [48] of Cq for GAPDH, RPLP0, and SF3A1 from the Cq for the gene of interest. Differences in expression between groups were calculated using the 2^−ΔΔCq method [54]. P-values were calculated based on two-tailed t-test, and P<0.05 was considered to indicate significance.

Search Tool for the Retrieval of Interacting Genes/Proteins v. 10 (STRING; http://string-db.org) linked to the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to recognize specific DNA repair pathways related to outcome that were significantly enriched and possible protein-protein interactions. A minimum of three counts and P<0.05 were considered to indicate the significant relevance of pathways.