How to Transfer RNA Gels
- 1). Gel staining--Gel staining is carried out prior to transfer to ensure that RNA is present in the gel and has migrated to the correct size and with sufficient separation. It is easily carried out before blotting by soaking in various nucleic acid-binding chemicals such as ethidium bromide. The gel can be photographed, subsequently and once this confirms the RNA's size and presence, the blot can be performed.
- 2). Preparation of gel--Transfer gel to an RNase-free container and soak in deionized water to rinse off all formaldehyde. Change several times as any remaining formaldehyde will interfere with the transfer of RNAs onto the membranes. The gel is also soaked in an alkaline hydrolysis buffer which breaks up longer RNA strands and facilitates subsequent transfer or larger molecules.
- 3). Cut a piece of sponge, twenty pieces of laboratory-grade C-fold paper towels, six pieces of Whatman 3MM filter paper, and nylon or nitrocellulose blotting membrane, very slightly larger (~0.5 cm) larger than the gel. Place the sponge in a deep container and soak with sodium citrate buffer.
Layer three filter papers on top of the sponge, ensuring that they also become soaked with buffer. Overlay the gel on top of the filter paper and use a glass or plastic pipette tip to roll out any bubbles present between the gel and the filter paper. The bubbles will prevent the RNA from transferring across to the membrane. Wrap a ribbon of plastic wrap carefully around the sides of the gel to ensure the electric current passes through the gel rather than run straight out of its edges.
Prepare the membrane by soaking in a large container of distilled water or sodium citrate buffer. Place the wet membrane over the exposed surface of the gel. If air bubbles are present, remove them by rolling out with a pipette tip. Top the sandwich with sodium citrate buffer, and layer the sandwich with the remaining three pieces of filter paper. Finish the sandwich with about 4 cm of filter papers. Weight the stack down overnight and ensure sufficient buffer is present to stop it drying out. - 4). Hybridization with labeled probe -- This is carried out the next day to detect the RNAs that have transferred onto the membrane. The hybridized probe is then subjected to autoradiography to reveal its location on the membrane.
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