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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.