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Using DNA aptamer probe for immunostaining of cancer frozen tissues.

ABSTRACT: Tissue immunostaining is critically important in clinical applications, and antibodies have been used extensively as the molecular probes. Recently, aptamer, as a new class of probes, have attracted much attention for their potential clinical and research value. Epithelial cell adhesion molecule (EpCAM) is a specific biomarker which is overexpressed in many cancers of epithelial origin. Here, a DNA-based EpCAM aptamer SYL3C is reported as a probe for the immunostaining of frozen and paraffin-embedded sections of colorectal cancer tissues. Commercialized EpCAM antibodies were also used as a standard control. EpCAM aptamer SYL3C specifically recognized and immunostained cancer nests of colorectal tumor sections, but it neither reacted with background cells within tumor sites nor exhibited cross-reaction to the benign lesions or inflammation of colorectal tissues. No cross-linking to EpCAM-negative malignant tumor sections occurred. Compared with standard antibody staining, our EpCAM aptamer SYL3C protocol is simpler to implement with a shorter reaction time. Moreover, SYL3C can specifically bind with either frozen or paraffin-embedded tissue sections. Since the histopathology of frozen tissue is closer to that of fresh tissue and since frozen sections can be produced more quickly than paraffin-embedded sections, SYL3C immunostaining of frozen sections is a quick protocol that is easy to implement.

SUBMITTER: Pu Y

PROVIDER: S-EPMC4318623 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Using DNA aptamer probe for immunostaining of cancer frozen tissues.

Pu Ying Y Liu Zhenxu Z Lu Yi Y Yuan Peng P Liu Jun J Yu Bo B Wang Guodong G Yang Chaoyong James CJ Liu Huixia H Tan Weihong W

Analytical chemistry 20150116 3

Tissue immunostaining is critically important in clinical applications, and antibodies have been used extensively as the molecular probes. Recently, aptamer, as a new class of probes, have attracted much attention for their potential clinical and research value. Epithelial cell adhesion molecule (EpCAM) is a specific biomarker which is overexpressed in many cancers of epithelial origin. Here, a DNA-based EpCAM aptamer SYL3C is reported as a probe for the immunostaining of frozen and paraffin-emb ...[more]

PMID: 25536018

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Project description:Molecular subtyping of breast cancer is of considerable interest owing to its potential for personalized therapy and prognosis. However, current methodologies cannot be used for precise subtyping, thereby posing a challenge in clinical practice. The aim of the present study is to develop a cell-specific single-stranded DNA (ssDNA) aptamer-based fluorescence probe for molecular subtyping of breast cancer. Methods: Cell-SELEX method was utilized to select DNA aptamers. Flow cytometry and confocal microscopy were used to study the specificity, binding affinity, temperature effect on the binding ability and target type analysis of the aptamers. In vitro and in vivo fluorescence imaging were used to distinguish the molecular subtypes of breast cancer cells, tissue sections and tumor-bearing mice. Results: Six SK-BR-3 breast cancer cell-specific ssDNA aptamers were evolved after successive in vitro selection over 21 rounds by Cell-SELEX. The Kd values of the selected aptamers were all in the low-nanomolar range, among which aptamer sk6 showed the lowest Kd of 0.61 ± 0.14 nM. Then, a truncated aptamer-based probe, sk6Ea, with only 53 nt and high specificity and binding affinity to the target cells was obtained. This aptamer-based probe was able to 1) differentiate SK-BR-3, MDA-MB-231, and MCF-7 breast cancer cells, as well as distinguish breast cancer cells from MCF-10A normal human mammary epithelial cells; 2) distinguish HER2-enriched breast cancer tissues from Luminal A, Luminal B, triple-negative breast cancer tissues, and adjacent normal breast tissues (ANBTs) in vitro; and 3) distinguish xenografts of SK-BR-3 tumor-bearing mice from those of MDA-MB-231 and MCF-7 tumor-bearing mice within 30 min in vivo. Conclusion: The results suggest that the aptamer-based probe is a powerful tool for fast and highly sensitive subtyping of breast cancer both in vitro and in vivo and is also very promising for the identification, diagnosis, and targeted therapy of breast cancer molecular subtypes.

Dataset Information

Using DNA aptamer probe for immunostaining of cancer frozen tissues.

Publications

Using DNA aptamer probe for immunostaining of cancer frozen tissues.

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