Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

Summary

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

Abstract

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

Introduction

Extracellular non-coding small RNAs (sRNAs) represent a new class of disease biomarkers and potential therapeutic targets and likely facilitate cell-to-cell communication¹. The most widely studied type of sRNA are microRNAs (miRNA) which are approximately 22 nts in length and are processed from longer precursor forms and primary transcripts². miRNAs have been demonstrated to post-transcriptionally regulate gene expression through suppression of protein translation and induction of mRNA degradation². Nevertheless, miRNAs are just one of many types of sRNAs; as sRNAs can be cleaved from parent tRNAs (tRNA-derived sRNAs, tDR), small nucle....

Protocol

1. HDL Purification (~ 5.5 days)

1. Density-Gradient Ultracentrifugation (DGUC, ~ 5 days)
   1. Add 90 μL of 100x anti-oxidants to 9 mL of plasma isolated from fresh venous blood.
   2. Adjust plasma density with KBr from 1.006 g/mL to 1.025 g/mL by adding 0.251 g KBr to 9 mL of plasma from Step 1.1.1 (0.0278 g/mL KBr plasma). Rock the plasma until all the salt is dissolved at room temperature and transfer to ultracentrifuge tubes and ensure all bubbles rise to the top.
   3. Bend the tip of an 18-G needle to 90° 1 cm from the tip, bevel side facing up. Using a syringe and the 18-gauge needle, carefully place 3 mL of overla....

Results

This protocol is a series of established methods linked together to allow for the quantification of sRNAs on highly pure HDL by high-throughput sequencing or real-time PCR (Figure 1). To demonstrate the feasibility and impact of this protocol, HDL was purified from human plasma by the tandem DGUC and FPLC method. Collected FPLC fractions corresponding to HDL (by cholesterol distribution) were concentrated and total RNA was isolated from 1 mg of HDL (total protein). sRNA l.......

Discussion

This protocol is designed to quantify miRNAs and other sRNAs by high-throughput sequencing or real-time PCR on highly pure HDL. As with any approach, special considerations should be given to each step in the process of purifying HDL and RNA and then quantifying sRNAs. This protocol is designed for projects starting with ≥ 2 mL of plasma. Nevertheless, high quality RNA analyses can successfully be completed with HDL purified from as little as 80 µL of human or mouse plasma using affinity chromatography; howeve.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ultracentrifuge	Beckman Coulter	A99839	Optima XPN-80
Ultracentrifuge Rotor	Beckman Coulter	331362	SW-41Ti
AKTA Pure FPLC System	GE Healthcare	29018224
3x FPLC Superdex 200 Increase Columns In-line	GE Healthcare	28990944	10/300 gl
SynergyMx	BioTek Instruments	7191000
Tabletop centrifuge	Thermo Scientific	75004525	Sorvall ST40R
Refrigerated centrifuge	Eppendorf	22629867	5417R (purchased through USA Scientific)
Microfuge	USA Scientific	2631-0006
PippenPrep	Sage Science	PIP0001
2100 Bioanalyzer	Agilent	G2938B
High Sensitivity DNA Assay	Agilent	5067-4626
Sequencing Library qPCR Quantification Kit	Illumina	SY-930-1010
ProFlex Thermal Cycler	Applied Biosystems	4484073
QuantStudio 12k Flex	Applied Biosystems	4471134
EpMotion Robot	Eppendorf	960000111	5070
Ultra-clear centrifuge tubes	Beckman Coulter	344059
Potassium Bromide	Fisher Chemicals	P205-500
15 mL conical tube	Thermo Scientific	339650
Micro-centrifugal filters 0.45 µm	Millipore	UFC30HV00
Micro-centrifugal filters 0.22 µm	Millipore	UFC30GV00
miRNAEasy Total RNA Isolation Kits	Qiagen	217004
Total Cholesterol colormetric kit	Cliniqa (Raichem)	R80035
10,000 m.w. cut-off centrifugation filter	Amicon	UFC801024	purchased through Millipore
PCR strip tubes	Axygen	PCR-0208-C	purchased through Fisher
microRNA RT kit	Life Technologies	4366597	For 1000 reactions
PCR master mix	Life Technologies	4440041	50 mL bottle
Pierce BCA kit	Thermo Scientific	23225
Clean and Concentrator Kit	Zymo	D4014
Dialysis tubing	Spectrum Labs	132118	purchased through Fisher
bcl2fastq2	Illumina	n/a	Software
Cutadapt	https://github.com/marcelm/cutadapt	n/a	Software
NGSPERL	github.com/shengqh/ngsperl	n/a	Software
CQSTools	github.com/shengqh/CQS.Tools	n/a	Software
Bowtie 1.1.2	http://bowtie-bio.sourceforge.net	n/a	Software
GeneSpringGX13.1.1	Agilent	n/a	Software