Sybr green quantitect primers assay

We used snapshot mRNA extracted from peripheral blood mononuclear cells (PBMC) sampled at the same time-of-day (09:00 a.m.). Total RNA from patients and controls was extracted using the RNeasy^® Mini Kit (QIAGEN) and subsequently digested by DNase I. cDNA was synthesized from 100 ng total RNA with Quantifast RT-PCR kit (QIAGEN). For real-time PCR, we used the Human QuantiTec Primers Assay (SYBR Green QuantiTect Primers Assay; QIAGEN) (Supplementary Table S1). All qPCRs were performed in a 10 μL final volume, with three replicates per sample. Reactions were set up in 96-well plates using a 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Expression levels of the target genes were normalized using the housekeeping control gene GAPDH. The mRNA amount of each target gene relative to control gene was calculated through the comparative Ct method (i.e., the 2(−ΔΔCt) method).

To assess the differential expression of the cryptochrome genes in CRC specimens and matched normal mucosa, quantitative real-time PCR (q-PCR) assay was performed by using CRY1 (QT00025067), CRY2 (QT00094920), ARNTL (QT00011844), WEE (QT00038199) and c-MYC (QT00035406) Human QuantiTec Primers Assay (SYBR Green QuantiTect Primers Assay; QIAGEN). All qPCRs were performed in a 25-μl final volume, with three replicates per sample, by using QuantiFast SYBR Green PCR kit (QIAGEN) and run in an ABI PRISM® 7700 Sequence Detection System (Applied Biosystems). The data were analyzed using the default and variable parameters available in the SDS software package (version 1.9.1; Applied Biosystems). GAPDH housekeeping control gene was used to normalize target gene expression levels and the mRNA amount of each target gene relative to GAPDH was calculated through the comparative Ct method, also called the 2(−ΔΔCt) method. Two biological replicates were each assayed in triplicate and results were expressed as mean ± standard deviation (SD).