New Technique Reduces Loss of MicroRNAs from Biological Specimens

Usually biological specimens such as tissues or cells are treated with a cross linking agent such as formaldehyde to fix molecules in place. The agent locks the specimen's macromolecules into a type of minute scaffolding, but as explained by first author Dr. John Pena, assistant professor of molecular biology at the Rockefeller University (New York, NY, USA), "Because microRNAs are so small, they come out of the tissue. They just fall through the scaffold."

Dr. Pena and his colleagues reported in the January 11, 2009, online edition of the journal Nature Methods that the loss of microRNAs from formaldehyde-fixed tissues could be prevented by subsequent fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). This treatment irreversibly immobilized the microRNA at its 5' phosphate end.

They tested this method on more than 130 microRNAs in an assortment of tissues including heart, liver, muscle, and brain. Results showed that retention of microRNAs by tissues treated with formaldehyde and EDC was consistently greater than in tissues treated with formaldehyde alone.

"If microRNAs are going to be used as a diagnostic tool, we need a reliable way to measure them in normal and disease tissue, regardless of where or to what degree they are expressed," said Dr. Pena. "So that is what we did: We went back and examined and questioned every single step of this protocol to see where we could optimize it. In order to move forward, we had to go to the basics."

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