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. 2018;1740:139-153.
doi: 10.1007/978-1-4939-7652-2_11.

Isolation of Plasma Lipoproteins as a Source of Extracellular RNA

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Free PMC article

Isolation of Plasma Lipoproteins as a Source of Extracellular RNA

Kang Li et al. Methods Mol Biol. .
Free PMC article

Abstract

Plasma lipoproteins are essential vehicles of lipid distribution for cellular energy and structural requirements as well as for excretion of lipid excess. Imbalances in lipoprotein metabolism are known to contribute to metabolic diseases ranging from vascular inflammation and atherosclerosis to obesity and diabetes. The lipid and protein cargo carried by lipoprotein subclasses have long been the focus of studies exploring the contribution of plasma lipoproteins in health and in metabolic disorders. More recent studies have revealed the presence of noncoding RNA as a new form of cargo carried by plasma lipoproteins. Lipoprotein-associated microRNAs have been identified to distribute differentially among plasma lipoprotein subclasses and contribute to cellular signaling. These findings highlight plasma lipoprotein-associated RNA as a potential source of biological signaling and warrant a renewed interest in the study of plasma lipoprotein biology. This chapter describes principles and methods based on density ultracentrifugation and size exclusion chromatography for the isolation of plasma lipoproteins as a source of extracellular RNA.

Keywords: Extracellular RNA; FPLC chromatography; HDL; LDL; Lipoprotein; Sequential density ultracentrifugation; VLDL.

Figures

Figure 1.
Figure 1.. Relationship between the densities and size among plasma lipoproteins
VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein
Figure 2.
Figure 2.. Experimental workflow of sequential density Ultracentrifugation (SD-UC) for lipoprotein fractionation
(1) Collect and pool blood and centrifuge 2,000 x g for 10 min to pellet blood cells; (2) Recover cell-free plasma and centrifuge 15,000 x g for 30 min to pellet platelets (PLT) and float chylomicrons (CM); (3) Collect PLT-free, CM-free plasma and add KBr to adjust its density to 1.063 g/mL; transfer it into a ultracentrifugation tube and fill the tube with saline that has been adjusted to the same density; seal the tubes and centrifuge 100,000 x g for 20 hours to float the VLDL and LDL; (4) Cut the top of the tube to recover VLDL/LDL and add KBr to adjust the density of the remaining liquid from 1.063 g/mL to 1.21 g/mL; transfer it to a fresh ultracentrifugation tube and fill the tube with saline (density=1.21 g/mL); seal the tubes and centrifuge 100,000 x g for 18 hours to float the HDL fraction; (5) cut the top of the tube to collect HDL.
Figure 3.
Figure 3.. Cholesterol profiles of total plasma and plasma lipoproteins separated by SD-UC and FPLC
VLDL/LDL and HDL were separated from plasma (6 mL) by sequential density ultracentrifugation. (A) The cholesterol content before and after isolation were determined; (B) Cholesterol profiles of total FPLC-fractionated plasma (200 μL), VLDL/LDL (100 μL) and HDL (75 μL).
Figure 4.
Figure 4.. Apolipoprotein distribution of total plasma and plasma lipoproteins separated by SD-UC and FPLC
VLDL/LDL and HDL were separated from plasma (6 mL) by sequential density ultracentrifugation. (A) SDS-PAGE analysis of total plasma (3 μL), VLDL/LDL (10 μL) and HDL (10 μL). (B, C and D) SDS-PAGE analysis of FPLC fractions of 200 μL plasma (B), 100 μL VLDL/LDL (C) and 75 μL HDL (D). For each fraction, 90 μL sample was loaded. P: plasma; V/L: VLDL/LDL; H: HDL.
Figure 5.
Figure 5.. Lipoprotein RNA Characterization by Agilent Bioanalyzer
Representative images of Agilent RNA 6000 Pico chip (A, B) and Small RNA chip (C, D) assays of total RNA isolated from plasma VLDL/LDL and HDL fractions isolated by sequential density ultracentrifugation.

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