Research note: methodology for high-quality RNA extraction from poultry whole blood for further gene expression analysis

Abstract

1. There are no published methods for RNA isolation from avian whole blood where nucleated red blood cells prevent the use of established mammalian protocols. The aim of this study was therefore to develop a protocol for total RNA extraction using avian whole blood by defining the effect of anticoagulants and sample purification protocols on RNA yield and quality.

2. Blood collections from the cutaneous ulnar or medial metatarsal veins of birds yielded adequate blood volume (2–3 ml) draws. The experiment was a 2 × 2 × 3 factorial arrangement of treatments, with two levels of DNase (0 and TURBO DNA-free Kit), two levels of Cleanup (0 and RNeasy MinElute Cleanup Kit), and three anticoagulants (no anticoagulant, EDTA, or sodium citrate).

3. RNA was isolated successfully by adding TRIzol LS to 0.25 ml of chicken whole blood at 3:1 ratio. From 0.125 ml of avian whole blood, 2–3 µg of RNA with RNA integrity number values of 7.75 was successfully isolated with the TRIzol LS extraction and an RNeasy MinElute Cleanup Kit.

4. This reliable protocol can be used to extract high yield and quality of total RNA from a small amount of whole blood.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical assistance provided by the staff and students of the University of Alberta Poultry Research Centre, Edmonton, Alberta.

FUNDING

Financial support for this study was provided by Alberta Livestock and Meat Agency, Edmonton, Alberta [Grant number 2011F121R].