RT-PCR kit for detection of gene expression in enriched tumor cells associated with epithelial-mesenchymal transition and cancer cell stemness. For isolation of CTCs and subsequent analysis of EMT, cancer cell stemness and breast cancer characteristics. The Kit is designed for 12 EMT marker determinations

Features

Efficient isolation and detection of CTCs from whole blood

Molecular characterization of CTCs

High specificity and sensitivity

Capturing of EMT and stem-like cells

Product Details

AdnaTest EMT-2/StemCell  is used for the analysis of EMT (epithelial-mesenchymal transition) and cancer cell stemness characteristics in immunomagnetically-enriched tumor cells. AdnaTest EMT-2/StemCellSelect allows enrichment of circulating tumor cells from peripheral blood, and AdnaTest EMT-2/StemCellDetect allows subsequent analysis of EMT-2/StemCell-associated gene expression. The analysis of breast, colon, prostate and ovarian cancer-associated genes is also possible. The test requires the enrichment of CTCs from 5 ml blood followed by reverse transcription cDNA synthesis using the AdnaTest EMT-2/StemCellDetect and AdnaWash (a specialized wash buffer that reduces leukocyte cross-reactions prior to the multiplex PCR assay).

Performance

AdnaTest EMT-2/StemCellSelect allows the enrichment of circulating tumor cells from peripheral blood and AdnaTest EMT-2/StemCellDetect allows for the subsequent analysis of EMT-2/StemCell-associated gene expression. The specificity of the detection is 90%. Using EMT-2 markers in spiking experiments, 20 tumor cells in 5 ml of whole blood are detected at a recovery rate of at least 70%. Using StemCell markers in spiking experiments, 10 tumor cells in 5 ml of whole blood are detected at a recovery rate of at least 70%.

Principle

The AndaTest EMT/StemCell allows molecular characterization of CTCs by combining the AdnaTest EMT-2/StemCellSelect with the AdnaTest EMT-2/StemCellDetect using the Combination of Combinations Principle (COCP). The AdnaTest EMT-2/Stem-CellSelect is a highly specific immunomagnetic cell selection system for enrichment of circulating tumor cells from peripheral blood. The AdnaTest EMT-2/StemDetect allows sensitive analysis of cancer-associated gene expression in immunomagnetically-enriched tumor cells by reverse transcription and PCR. The test requires the enrichment of CTC from 5 ml blood followed by reverse transcription cDNA synthesis using the AdnaTests EMT-2/StemcellDetect and AdnaWash (a specialized wash buffer that reduces leukocyte cross-reactions prior to the multiplex PCR assay). The kit contains primer-mix to amplify c-DNA target fragments of Twist, PI3Kα, Akt2 and a positive control (actin). Analysis of breast, colon, prostate and ovarian cancer-associated genes is also possible.

Procedure

The AdnaTest uses a two-step process (Select and Detect) to generate results within 5 hours. AdnaTest EMT-2/StemCellSelect 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 . In the second step, the isolated mRNA is transcribed into cDNA and can be amplified using multiplex PCR . AdnaTest EMT-2/StemCellDetect 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 EMT tumor markers.

The Targets

TWIST

Basic helix-loop-helix (bHLH) transcription factors have been implicated in cell lineage determination and differentiation. The protein encoded by this gene is a bHLH transcription factor and shares similarity with another bHLH transcription factor, Dermo1. It is highly expressed in placental tissue and in adult mesodermally-derived tissues. Twist binds to the E-box elements of the Akt2 promoter, enhancing its transcriptional activity. It's likely that this transcription factor is related to the EMT phenomenon in cancer cells.

Pi3K/Akt

Pi3K activates the Akt1 and Akt2 Ser/Thr kinase. Activated Akt is responsible for proliferation and has anti-apoptotic function (inhibition of caspase 9 activity). There are several possibilities that can cause the Pi3K/Akt (mTOR) signal cascade to become unregulated in cancer. One possibility is the loss of PTEN activity, which is a regulator of Pi3K that leads to a permanent activation of the pathway. Since the Pi3K/Akt pathway is also activated via tyrosine kinases it may be affected in case of EGFR overexpression/mutation and HER2 overexpression. Another activator is the IGF receptor, which has been described in breast cancer to potentially shortcut ER dependency for proliferation if overexpressed.

ALDH1

Utilizing in vitro and in vivo experimental systems, it was shown (Ginestier et al 2007) that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase 1 activity (ALDH1) have stem/progenitor properties. Overexpression of ALDH1 in tissue of primary breast tumors correlates significantly with poor prognosis. In breast carcinomas, high ALDH1 activity leads to increased self-renewal and tumors that mimic the heterogeneity of the parental tumor.

AdnaTest EMT-2/StemCellDetect contains Oligo (dT)25-coated beads for the isolation of mRNA from the lysate of pre-enriched tumor cells. Reverse transcription results in cDNA, which is subsequently used as template for tumor cell detection and characterization by multiplex PCR. The PrimerMix EMT-1 allows the amplification of three EMT-related genes and one control gene. The PrimerMix StemCell amplifies ALDH1, which is accepted as tumor stem-cell marker. Breast cancer associated gene expression can be determined using the PrimerMix BreastDetect. Taken together the different primer-mixes generate the following fragments:

PrimerMix EMT-2

 Target gene                      PCR fragment size (bp)

 PI3Kα                                       551

 AKT-2                                       309

 TWIST1                                       201

 Actin (internal PCR control)       120

PrimerMix Stem Cell

 Target gene PCR fragment size (bp)

 ALDH1                    161

Optionally, the analysis of breast, colon, prostate and ovarian cancer-associated genes is possible. The PrimerMix Colon Detect allows the amplification of three tumor-associated antigens and one control gene. The primers generate fragments of the following sizes:

PrimerMix Colon Detect

 Target gene             PCR fragment size (bp)

 GA733-2                                   395

 CEA                                           231

 EGFR                                   163

 Actin (Internal PCR control)   120

The PrimerMix Breast Detect allows the amplification of three tumor-associated antigens and one control gene. The primers generate fragments of the following sizes:

PrimerMix Breast Detect

 Target gene         PCR fragment size (bp)

 GA733-2                                  395

 Muc-1                                  299

 Her-2                                  265

 Actin (Internal PCR control) 120

The PrimerMix Prostate Detect allows the amplification of three tumor-associated antigens and one control gene. The primers generate fragments of the following sizes:

PrimerMix Prostate Detect

 Target gene            PCR fragment size (bp)

 PSMA                                    449

 PSA                                            357

 EGFR                                    163

 Actin (Internal PCR control)    120

Importantly, the AdnaTest is an open platform that allows the profiling of additional custom selected mRNA targets for comphrehensive biomarker discovery.

Applications

Isolation of CTCs from whole human blood

Molecular characterization of CTCs

Biomarker discovery

mRNA profiling

Liquid biopsy