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To verify the kits, 1, 5,10, and 50 MDA-468 or the SKBR3 cells were directly spiked into lysis buffer followed by qualitative PCR amplifying EpCAM, EGFR, ERBB2, KRT19, and ACTB transcripts.
The KRT19 transcripts have been widely used to identify and study CTCs in cancer patients (5, 6).
The RNA sample with the higher integrity (C71A) recorded lower Cqs for KRT19 (keratin 19), UBC#1, and HMBS (hydroxymethylbilane synthase), but not for TP53I3 (tumor protein p53 inducible protein 3) and GAPDH (see online Supplemental Tab 12).
5] Human genes: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CDH1, cadherin 1, type 1, E-cadherin (epithelial); CTNNB, catenin (cadherin-associated protein), [beta]1, 88 kDa; MAX, MYC associated factor X; CDK2, cyclin-dependent kinase 2; RBL1, retinoblastoma-like 1; MYC, v-myc avian myelocytomatosis viral oncogene homolog; UBC, ubiquitin C; KRT19, keratin 19; HMBS, hydroxymethylbilane synthase; TP53I3, tumor protein p53 inducible protein 3.
KRT19 EXPRESSION AND SOX17 METHYLATION IN CTCs AND cfDNA
We evaluated KRT19 mRNA expression in all matched EpCAM-positive immune-magnetically isolated CTC fractions, because this epithelial marker has been extensively used to verify the presence of CTCs (1, 4, 5, 20, 21).
Our group developed an RT-qPCR assay for KRT19 mRNA (36, 37) and evaluated both its analytical and diagnostic sensitivity and its specificity and clinical potential for the molecular detection of occult carcinoma cells in peripheral blood of breast cancer patients (10-13).
A higher rate of positive samples was observed with the use of a combined RT-qPCR approach for KRT19 and SCGB2A2 (mammaglobin), which suggests that this approach is currently the most analytically sensitive technique for detecting CTCs.
cDNA synthesis was performed using the SuperScript[TM] First-Strand Synthesis System (Invitrogen) and the cDNA was used for KRT19 expression studies in CTCs as described (31).
0) for data analysis and for the evaluation of the significance of differences between groups, and Fisher exact test for the evaluation of the correlation between methylation of each gene in respect to the patient's characteristics and DNA methylation of each gene in respect to KRT19 gene expression.
By using a multimarker assay for CTC in early breast cancer, we have shown that CTCs positive for KRT19 (keratin 19; also known as CK19), SCGB2A2 (secretoglobin, family 2A, member 2; also known as MGB1, mammaglobin A), and ERBB2 are associated with shorter disease-free survival (6).
The genes selected are established markers for CTCs: KRT19, a specific epithelial marker of prognostic significance (3-7, 9); SCGB2A2, a specific marker for mammary gland (6); ERBB2, which gives important information about response to therapy (12, 13); TWIST1 [twist homolog 1 (Drosophila)], a marker of epithelial-to-mesenchymal transition; and MAGEA3 (melanoma antigen family A, 3), the expression of which correlates with metastasis.
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