研究报告：从家禽全血中提取高质量 RNA 以进一步进行基因表达分析的方法。

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

a Department of Agricultural, Food and Nutritional Science , University of Alberta , Edmonton , AB , Canada T6G 2P5.

Br Poult Sci. 2014;55(2):194-6. doi: 10.1080/00071668.2014.888397.

Abstract

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.

摘要

目前尚无从禽类全血中提取 RNA 的方法，因为有核红细胞会阻止使用已建立的哺乳动物方案。因此，本研究的目的是通过定义抗凝剂和样品纯化方案对 RNA 产量和质量的影响，开发一种使用禽类全血提取总 RNA 的方案。
从鸟类的皮肤尺侧或内侧跖静脉采集的血液可获得足够的采血量（2-3 毫升）。该实验是一个 2×2×3 析因设计的处理安排，有两种水平的 DNase（0 和 TURBO DNA-free 试剂盒）、两种水平的清洁（0 和 RNeasy MinElute 清洁试剂盒）和三种抗凝剂（无抗凝剂、EDTA 或柠檬酸钠）。
通过将 TRIzol LS 以 3:1 的比例加入 0.25 毫升鸡全血中，可以成功分离 RNA。从 0.125 毫升禽类全血中，使用 TRIzol LS 提取和 RNeasy MinElute 清洁试剂盒成功分离到 2-3 µg 的 RNA，RNA 完整性数值为 7.75。
该可靠的方案可用于从小量全血中提取高产率和高质量的总 RNA。