Nucleic Acid Extraction and Detection Across Two-Dimensional Tissue Samples
Armani, Michael Daniel
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Visualizing genetic changes throughout tissues can explain basic biological functions and molecular pathways in disease. However, over 90% of mammalian messenger RNA (mRNA) is in low abundance (<15 copies per cell) making them hard to see with existing techniques, such as in-situ hybridization (ISH). In the example of diagnosing cancer, a disease caused by genetic mutations, only a few cancer-associated mRNAs can be visualized in the clinic due to the poor sensitivity of ISH. To improve the detection of low-abundance mRNA, many researchers combine the cells across a tissue sample by taking a scrape. Mixing cells provides only one data point and masks the inherent heterogeneity of tissues. To address these challenges, we invented a sensitive method for mapping nucleic acids across tissues called 2D-PCR. 2D-PCR transfers a tissue section into an array of wells, confining and separating the tissue into subregions. Chemical steps are then used to free nucleic acids from the tissues subregions. If the freed genetic material is mRNA, a purification step is also performed. One or more nucleic acids are then amplified using PCR and detected across the tissue to produce a map. As an initial proof of concept, a DNA map was made from a frozen tissue section using 2D-PCR at the resolution of 1.6 mm per well. The technique was improved to perform the more challenging task of mapping three mRNA molecules from a frozen tissue section. Because the majority of clinical tissues are stored using formalin fixation and not freezing, 2D-PCR was improved once more to detect up to 24 mRNAs from formalin-fixed tissue microarrays. This last approach was used to validate genetic profiles in human normal and tumor prostate samples faster than with existing techniques. In conclusion, 2D-PCR is a robust method for detecting genetic changes across tissues or from many tissue samples. 2D-PCR can be used today for studying differences in nucleic acids between tumor and normal specimens or differences in subregions of the brain.