RNA extraction from milk for HPAI surveillance
William C. Vuyk, Andrew Lail, Isla Emmen, David O'Connor
Disclaimer
Abstract
This is an RNA extraction method that isolates HPAI RNA segments from commercially-available milk samples. The protocol involves processing with a MagMax RNA extraction kit on a Kingfisher instrument. Then, the samples are concentrated using a Zymo Clean and Concentrate kit. The total RNA yields should be suitable for performing qPCR. After trying other methods involving centrifugation, filtration, and trizol, we determined this method to be the easiest and most effective.
Steps
RNA extraction
Use the MagMax Wastewater Ultra x96 kit1 (or comparable magnetic bead isolation kit2-5) on the ThermoFisher Kingfisher Apex (or comparable instrument). For each sample, load 400 uL of pasteurized milk or heavy whipping cream into the binding plate.
Elute in 50 uL of elution solution or nuclease-free water.
Optional Sample Concentration and Cleaning
Pool the eluate from 6 extraction replicates of the same sample.
Take 200 uL forward into the DNase treatment. Using ThermoFisher TurboDNase, combine 200 uL of pooled eluate, 22 uL 10X TurboDNase buffer, and 2 uL TurboDNase.
Incubate at 37 C for 30 minutes.
Use a Zymo RNA Clean and Concentrator-5 kit7 to clean and concentrate the sample. Elute this into 30 uL final volume of nuclease-free water for downstream analysis.
