PCR expression analysis of hormone receptor genes for estrogen and progesterone in enriched tumor cells
The AndaTest ER/PR-Detect is an extension to AdnaTest BreastCancer and allows the molecular characterization of CTCs for estrogen and progesterone gene expression. The AdnaTest BreastCancerSelect is a highly specific immunomagnetic cell selection system for enrichment of circulating tumor cells from peripheral blood. After immunomagnetically enrichment of circulating breast cancer tumor cells, AdnaTest BreastCancerDetect allows cDNA generation and AdnaTest ER/PR-Detect allows for sensitive analysis of estrogen and progesterone gene expression.
The AdnaTest ER/PR is a highly sensitive RT-PCR for the detection of hormone receptor-specific mRNA markers. The specificity of the detection is 90%. In spiking experiments, 5 tumor cells in 5 ml of whole blood were detected at a recovery rate of at least 90%.
AdnaTest technology uses an optimized combination of antibodies for cell selection in 5 ml whole blood and a combination of tumor-associated markers for the detection of tumor cells: EpCAM, CA15-3 (Muc1) and presence of Her2 receptors (optional detection of estrogen and progesterone receptors by AdnaTest ER/PR-Detect).
The AdnaTest uses a two-step process (Select and Detect) to generate results within 5 hours (see figures “AdnaTest workflow”). AdnaTest BreastCancerSelect enables the immunomagnetic enrichment of tumor cells from whole blood via epithelial- and tumor-associated antigens. Antibodies against epithelial- and tumor-associated antigens are conjugated to magnetic beads for labeling of tumor cells. Labeled cells are extracted by a magnetic particle concentrator (AdnaMag-L and AdnaMag-S), lysed and mRNA is then purified from these lysates (see figures “AdnaTest Select – Immunomagnetic cell selection with multiple tumor-associated antibodies labeled to magnetic beads”). In the second step, the isolated mRNA is transcribed into cDNA and can be amplified using multiplex PCR (see figures “AdnaTest Detect – Multiplex PCR of various cancer-associated tumor markers”). AdnaTest BreastCancerDetect contains Oligo (dT) 25-coated beads for the isolation of mRNA from the lysate of pre-enriched tumor cells. Reverse transcription results in cDNA that can subsequently be used as template for tumor cell detection and characterization by multiplex PCR. Multiplex PCR provides tumor-associated gene expression profiles for a variety of relevant tumor markers, ensuring that the selected cells are cancer cells. The PrimerMix ER/PR-Detect allows the amplification of the hormone receptor genes for estrogen, progesterone and one control gene (actin). The primers generate fragments of the following sizes:
Importantly, the AdnaTest is an open platform that allows the profiling of additional custom selected mRNA targets for comphrehensive biomarker discovery.
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