Formaldehyde affects nucleic acids by:
(1) blocking the base pairing needed for hybridization
(2) creating cross-links with other macromolecules which can reduce the amount that can be extracted
Factors affecting the quality of the extracted RNA:
(1) RNA extracted after formaldehyde adducts are removed is more labile and so may be hydrolyzed if exposed to strong treatments such as those used for antigen retrieval.
(2) Problems with initially tissue handling and processing may impact the quality of the extracted RNA.
RNA can be released from the effects of formaldehyde without excessive hydrolysis by:
(1) using a dilute buffer (phosphate, Tris, other) at pH 8 and
(2) incubating at 70°C for 30 minutes
where:
• Raising the temperature to 70°C results in a drop in buffer pH. A pH of 6 to 7 is desirable for demodification.
• Amines (present in Tris buffers and believed to act as a direct formaldehyde scavenger) are not required for good recovery.
The precise conditions for optimum recovery of intact RNA may vary with each laboratory.
The status of the RNA should be monitored for evidence of excessive hydrolysis.
