Analysis at the mRNA level of lymph node tissue has several advantages over protein level or cellular level analyses. Multiple biomarkers can easily be analyzed in the same RNA extract and a large volume of the lymph node (potentially the entire node) can be analyzed.
In the ColoNode® kit six target genes are analyzed. Four of the genes are expressed in colorectal cancer epithelial cells, namely CEACAM5, KLK6, SLC35D3 and MUC2, and one biomarker, POSTN, is expressed in tumor associated fibroblasts. None of the genes are expressed in immune cells making them suitable for analysis of lymph node samples. 18S rRNA is expressed in all cell types and is analyzed for normalization and as a positive control for total-RNA suitable for real-time, quantitative RT-PCR, i.e. the technology used in the ColoNode® kit.
CEACAM5 protein is a well‐established tumor marker in adenocarcinoma and CEACAM5 mRNA levels correlate well to number of tumor cells
High levels of KLK6 mRNA correlate to poor prognosis
Expression of SLC35D3 mRNA is associated with poor prognosis
- High levels of stromal POSTN mRNA correlate to poor prognosis
Lymph nodes harboring tumor cells with high levels of MUC2 mRNA is an indicator of good prognosis
The ColoNode® kit is designed as two triplex, one-step qRT-PCR assays that are run simultaneously, one reaction measuring CEACAM5, KLK6 and SLC35D3 and one reaction measuring POSTN, MUC2 and 18S rRNA. The kit performs the reverse transcription (RT), amplification (PCR) and quantitation of mRNA by qRT-PCR with two triplex reaction mixes. It contains two master mixes with primers and probes for the respective three target genes. It also contains standard samples for actual mRNA content determination (copies/µl) and negative control. Unknown lymph node samples, standards and negative control are analyzed in duplicate.
Specific reverse and forward primers have been constructed for each of the six target genes. They are placed in different exons of respective target gene. A sequence-specific dye-labeled probe is placed over the boundary between the two exons to make the assay RNA specific and thereby avoiding amplification of genomic DNA. Three different fluorescence dyes (FAM™, VIC™, NED™; Thermo Fisher Scientific Inc) are used to perform quantification of 3 target genes in a single reaction. Three genes are measured in half the number of wells on the plate and the other 3 genes are measured in the other half.
In each analysis, standard samples containing specific amounts of RNA-copies from each of the 6 target genes are analyzed simultaneously with the unknown samples and form six standard curves. The analyzed unknown samples get the actual gene quantity per µl calculated from the respective target gene standard curve.