54 - Tissue-Print Hybridization for Detecting RNA Directly

Abstract

Tissue printing is a simple method for detecting macromolecules blotted directly from the surfaces of severed organs onto nylon or nitrocellulose membranes. The blotting procedure produces an image of the cut surface of the tissues on the membrane. Macromolecules such as proteins, complex carbohydrates, and nucleic acids are fixed to the membrane. The retention of nucleic acids on the membrane allows the detection of RNAs by hybridization with either DNA or antisense RNA probes. For tissue printing, a dry nylon membrane is placed over a single layer of dry whatman paper or some other absorbent paper. Organs or organ sections are prepared for printing onto membranes by sectioning through the organ with a single- or double-edged razor blade. The freshly cut surfaces are pressed immediately to the nylon membrane or lightly blotted with kimwipes prior to blotting to the membrane. Tissue printing is performed by using firm pressure with the index finger above the sectioned organ for 30–120 sec. The quality of the tissue prints is evaluated by examining the printed nylon membrane under a UV light source. Under UV light, it is possible to observe whether any organ sections were crushed or distorted during blotting. It is found that the large organs of firm consistency such as coytledons, stems, and petioles are much easier to tissue print than small or less firm organs such as roots, leaves, or floral parts.