Construction and Clinical Validation of a Colorectal Cancer OPISV Prognostic Model Integrating EPHB2 and ZNF346: Potential Regulatory Role of the Axon Guidance Pathway
Construction and Clinical Validation of a Colorectal Cancer OPISV Prognostic Model Integrating EPHB2 and ZNF346: Potential Regulatory Role of the Axon Guidance Pathway
Tissue specimens were collected from surgically resected tumor samples of patients, snap-frozen at -80ì§¸C, and transferred in liquid nitrogen. This study was approved by the Ethics Committee. Total RNA was extracted from the samples using the [Insert Kit Name, e.g., TRIzol reagent / Qiagen RNeasy Mini Kit] according to the manufacturer's instructions. The concentration and purity of the RNA were determined by measuring the absorbance at 260 nm and 280 nm using a NanoDrop spectrophotometer (A260/A280 ratio). RNA integrity was assessed by agarose gel electrophoresis to verify the presence of intact 18S and 28S ribosomal RNA bands. The poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated.