May 10, 2024
RNA extraction from milk for HPAI surveillance
- 1University of Wisconsin-Madison
Protocol Citation: Andrew Lail, William C. Vuyk, Isla Emmen, David O'Connor 2024. RNA extraction from milk for HPAI surveillance. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn6obgk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 01, 2024
Last Modified: May 10, 2024
Protocol Integer ID: 99087
Keywords: Dairy, Milk, Influenza, Viral RNA, RNA Extraction
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.
Materials
MagMax Wastewater Ultra x96 kit
Zymo RNA Clean and Concentrator-5 kit
ThermoFisher TurboDNase kit
Heat block
Lab plasticware
Safety warnings
Performing this protocol with unpasteurized milk from an infected cow might expose the user to active flu virus. Please use pasteurized samples or take appropriate precautions.
RNA extraction
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
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.
Protocol references
1. MagMAXTM Wastewater Ultra Nucleic Acid Isolation Kit [Internet]. [cited 2024 May 10]. Available from:
https://www.thermofisher.com/order/catalog/product/A52606
2. Guidelines and minimum requirements for diagnosis of H5N1 HPAIV infection in cattle [Internet].
Istituto Zooprofilattico Sperimentale delle Venezie; 2024 Apr [cited 2024 May 10]. Report No.: Version 2024.04.10. Available from: https://www.izsvenezie.com/documents//reference-laboratories/avian-influenza/diagnostic-protocols/guidelines-diagnosis-h5n1-cattle.pdf
3. MagMAXTM-96 Viral RNA Isolation Kit [Internet]. [cited 2024 May 10]. Available from: https://www.thermofisher.com/order/catalog/product/AM1836
4. MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit [Internet]. [cited 2024 May 10]. Available from: https://www.thermofisher.com/order/catalog/product/A42356
5. MagMAXTM Microbiome Ultra Nucleic Acid Isolation Kit, with bead tubes [Internet]. [cited 2024 May 10]. Available from: https://www.thermofisher.com/order/catalog/product/A42358
6. TURBOTM DNase (2 U/μL) [Internet]. [cited 2024 May 10]. Available from: https://www.thermofisher.com/order/catalog/product/AM2238
7. ZYMO RESEARCH [Internet]. [cited 2024 May 10]. RNA Clean & Concentrator-5. Available from: https://www.zymoresearch.com/products/rna-clean-concentrator-5