Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.
Abstract
In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.
This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.
Materials
ELF-97
Life technology
E6604
50X Denhard't
Life technology
750018
t-RNA
Roche
109541
Reconstitute in DEPC water
Herring Sperm DNA
Promega
D1815
Roche Blocking
Roche
11096176001
RVC
Fisher
50-812-650
Before use spin down it at 16.1 RCF and take supernatant
Patel, Y., Lee, J. S., Chen, H. Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization. J. Vis. Exp. (112), e53928, doi:10.3791/53928 (2016).