Diagnostics kits for the detection of CTCs in solid tumors
One of the most suitable methods for the detection of circulating tumor cells (CTCs) in patients with solid tumors is a methodology based on the principle of real-time RT-PCR (real-time reverse transcription polymerase chain reaction). The basic is to detect the expression of epithelial tumor characteristics in compartments of mesenchymal origin. This is an indirect method which enables to prove tumor cells in compartments distant from primary tumor by detection of the marker or its overexpression. Complete RNA purified from patients' sample cells (native or fixed tissues, bone-marrow cells, blood, body fluids containing cell material) is transcribed by reverse transcriptase into complementary DNA (cDNA). Copy number of the examined marker (gene) in the sample is determined by the standardization curve using specific hydrolysis TaqMan probes in real-time RT-PCR. The real-time PCR is an enzymatic reaction using primers, a specific fluorescently labeled probe, and thermostable DNA-polymerase, which is monitored by a thermocycler in real-time and based on the detection of fluorescence variations. After adding standards to the reaction, and after creating the standardization curve, the amount of the specific sequence in the sample can be assessed. To every polymerase, a reaction buffer of suitable composition is added. An optimal concentration of magnesium ions should be specified for every new primer pair, new polymerase, and new thermocycler. It is advantageous to use DNA-polymerase with a so called „HotStart“, that must be activated by heating it up for about 10 to 15 minutes to 90-95 °C. The primers are short oligonucleotides (with a length of ca. 20 bp) that define the amplifed part of DNA; the specific hydrolisis TaqMan probe is a short oligonucleotide labeled with flourophore and a quencher. The primer setting temperature is approximately calculated from the nucleotide composition, and should be later verified by experiments when optimizing the amplification cycle.