<abstract xmlns="http://www.w3.org/1999/xhtml"><p>The goat (<italic>Capra hircus</italic>) is a perfect animal model for analyzing the transcriptome of milk somatic cells (MSCs), as sufficient numbers of somatic cells in goat milk, i.e., exfoliated epithelial cells, can be obtained using noninvasive methods. RNA integrity and purity are the first and most important parameters qualifying samples for transcriptomic tests and next-generation sequencing, as RNA quality influences experimental results. The aim of this study was to optimize a method for obtaining high-quality RNA from goat MSCs, irrespective of effects like breed, lactation stage, health status (e.g., with or without small ruminant lentivirus [SRLV] infection), or number of somatic cells. Milk samples were obtained from goats of two Polish breeds in various lactation stages and in different parities, and from goats infected and not infected with SRLV. Altogether, 412 MSC samples were examined: 206 using method A with fenozol and 206 using method B with QIAzol. Though the overall purity (measured as absorbance ratios at 260 nm/280 nm and 260 nm/230 nm) of the RNA material was comparable, the average yield of RNA isolated using method A was 11.9 µg, while method B’s average yield was 29.9 µg. Moreover, method B resulted in good quality RNA suitable for transcriptome analysis. Results were confirmed by RT-qPCR, using <italic>18S rRNA</italic> and <italic>RPLP0</italic> as the reference genes. The application of our modified treatment method was successful in obtaining high-integrity samples for transcriptomic or next-generation sequencing analysis. Using a 400 mL milk sample cooled in ice directly after milking, securing the cooling chain process from milking to MSC isolation, and applying method B to isolate RNA, we obtained good RNA quality irrespective of the goats’ breed, lactation stage, parity, milk yield, SRLV infection, and even milk yield and number of somatic cells in milk.</p></abstract>

The goat (Capra hircus) is a perfect animal model for analyzing the transcriptome of milk somatic cells (MSCs), as sufficient numbers of somatic cells in goat milk, i.e., exfoliated epithelial cells, can be obtained using noninvasive methods. RNA integrity and purity are the first and most important parameters qualifying samples for transcriptomic tests and next-generation sequencing, as RNA quality influences experimental results. The aim of this study was to optimize a method for obtaining high-quality RNA from goat MSCs, irrespective of effects like breed, lactation stage, health status (e.g., with or without small ruminant lentivirus [SRLV] infection), or number of somatic cells. Milk samples were obtained from goats of two Polish breeds in various lactation stages and in different parities, and from goats infected and not infected with SRLV. Altogether, 412 MSC samples were examined: 206 using method A with fenozol and 206 using method B with QIAzol. Though the overall purity (measured as absorbance ratios at 260 nm/280 nm and 260 nm/230 nm) of the RNA material was comparable, the average yield of RNA isolated using method A was 11.9 µg, while method B’s average yield was 29.9 µg. Moreover, method B resulted in good quality RNA suitable for transcriptome analysis. Results were confirmed by RT-qPCR, using 18S rRNA and RPLP0 as the reference genes. The application of our modified treatment method was successful in obtaining high-integrity samples for transcriptomic or next-generation sequencing analysis. Using a 400 mL milk sample cooled in ice directly after milking, securing the cooling chain process from milking to MSC isolation, and applying method B to isolate RNA, we obtained good RNA quality irrespective of the goats’ breed, lactation stage, parity, milk yield, SRLV infection, and even milk yield and number of somatic cells in milk.

An Optimized Method of RNA Isolation from Goat Milk Somatic Cells for Transcriptomic Analysis

Department of Food Hygiene and Public Health Protection, Faculty of Veterinary Medicine

Department of Physiology Sciences, Faculty of Veterinary Medicine

Department of Econometrics and Statistics, Faculty of Applied Informatics and Mathematics

Department of Preclinical Sciences, Faculty of Veterinary Medicine

Institute of Epidemiology and Veterinary Management, Faculty of Veterinary Medicine

Institute of Genetics and Animal Breeding

Annals of Animal Science

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

The goat (Capra hircus) is a perfect animal model for analyzing the transcriptome of milk somatic cells (MSCs), as sufficient numbers of somatic cells in goat...

An Optimized Method of RNA Isolation from Goat Milk Somatic Cells for Transcriptomic Analysis

Pubblicato online: 30 lug 2019

Pagine: 605 - 617

Ricevuto: 14 ott 2018

Accettato: 19 mar 2019

DOI: https://doi.org/10.2478/aoas-2019-0024

Parole chiave
goat, milk somatic cells, RNA isolation, transcriptomic analysis, RT-qPCR

© 2019 Joanna Pławińska-Czarnak et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

An Optimized Method of RNA Isolation from Goat Milk Somatic Cells for Transcriptomic Analysis

Pubblicato online: 30 lug 2019

Pagine: 605 - 617

Ricevuto: 14 ott 2018

Accettato: 19 mar 2019

DOI: https://doi.org/10.2478/aoas-2019-0024

Parole chiavegoat, milk somatic cells, RNA isolation, transcriptomic analysis, RT-qPCR

© 2019 Joanna Pławińska-Czarnak et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Parole chiave
goat, milk somatic cells, RNA isolation, transcriptomic analysis, RT-qPCR