2.4 Circulating
mRNA
Several circulating mRNA candidates have been studied for CRC
screening and prognosis after surgical resection [16–18]. mRNA
biomarkers in the blood, such as CEA, CK20, CK19, human
telomerase reverse transcriptase (hTERT), and guanylyl cyclase C
(GC-C), have all been examined in the perioperative setting
[17]. In most cases, the persistence of tumor-associated mRNA
within 24 h after tumor resection has been a strong predictor of
relapse [19]. In a meta-analysis of nine studies with CRC patients
undergoing curative surgery, CTC detection based on CEA, CK19,
and CK20 mRNA correlated with the development of hepatic
metastases and decreased disease-free survival [20]. Recently,
Rodia et al. utilized a novel Transcriptome Mapper (TRAM) to
identify some specific RNAs in the blood as the candidate mRNA
markers to detect CRC. After validation by qRT-PCR, the expres-
sion of genes including TSPAN8, LGALS4, COL1A2, and CEA-
CAM6 was found to be statistically different in the blood of
67 patients and 67 healthy controls [19]. However, circulating
mRNA also has its limitations. Extracellular mRNA is susceptible
to degradation by RNase in the plasma. Attempts to use RNase
inhibitors have been unsuccessful at protecting mRNA from degra-
dation [21]. Thus, the application of mRNA in cancer screening
and prognosis requires more investigation.3 Methods of Detecting Genomic Changes from cfDNA
Nowadays, there are various techniques to detect genetic altera-
tions in cfDNA, including real-time polymerase chain reaction
(RT-PCR), digital PCR, amplification protocols with magnetic
beads in oil emulsions (beads, emulsion, amplification, and mag-
netics (BEAMing)), next-generation sequencing (NGS), and mass
spectrometry (MS) genotyping. Currently there are few standar-
dized methods for processing cfDNA samples and analyzing the
results. Thus, it is necessary to establish evidence-based guidelines
in order to select the most reliable and cost-effective method for
clinical application. Here, these techniques will be described below.3.1 RT-PCR Three types of RT-PCR are routinely employed, based on the
probes utilized: TaqMan probes, TaqMan Detection Mutation
Assay, and Scorpion probes. TaqMan probes have a mutation detec-
tion limit of about 10%, whereas TaqMan Detection Mutation
Assay may detect mutations as low as 0.1% [22, 23]. These varia-
tions of sensitivity are due to the probe design: TaqMan probes
consist of a fluorophore covalently attached to the 5^0 -end of the
oligonucleotide probe and a quencher at the 3^0 -end, whereas Taq-
Man Detection Mutation Assay contains an allele-specific primer
and an MGB blocker oligonucleotide that suppresses the wild-type
background. Scorpion probes are covalently bound to a primer, a
48 Jun Li et al.