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Method Article

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

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DOI:

10.3791/54488

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November 28th, 2016

 ,  ,  ,  ,  ,  , 
1Department of Medicine, Vanderbilt University School of Medicine, 2Center for Quantitative Sciences, Vanderbilt University School of Medicine, 3Department of Cancer Biology, Vanderbilt University School of Medicine

* These authors contributed equally

In This Article

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 nuclear RNAs (sRNA-derived sRNAs, sndRNA), small nucleolar RNAs (snoRNA-derived sRNAs, snRNA), ribosomal RNAs (rRNA-derived sRNAs, rDR), Y RNAs (yDR), and other miscellaneous RNAs1. A few examples of these novel sRNAs have been reported to function similar to miRNAs; however, the biological functions of many of these sRNAs remains to be determined, although roles in gene regulation are likely3-6. Most interestingly, miRNAs and other sRNAs are stably present in extracellular fluids, including saliva, plasma, urine, and bile. Extracellular sRNAs are likely protected from RNases through their association with extracellular vesicles (EV), lipoproteins, and/or extracellular ribonucleoprotein complexes.

Previously, we reported that lipoproteins, namely high-density lipoproteins (HDL), transport miRNAs in plasma7. In this study, HDL were isolated using a sequential method of density-gradient ultracentrifugation (DGUC), fast-protein liquid chromatography (size-exclusion chromatography gel filtration, FPLC), and affinity chromatography (anti-apolipoprotein A-I (apoA-I) immunoprecipitation)7. Using both real-time PCR-based low-density arrays and individual miRNA assays, miRNA levels were quantified on HDL isolated from healthy and hypercholesterolemic subjects7. Using this approach, we were able to profile miRNAs and quantify specific miRNAs in highly pure HDL preparations. Since 2011, we have determined that although affinity chromatography enhances HDL purity, antibody saturation greatly limits yield, and can be cost-prohibitive. Currently, our protocol recommends a two-step sequential tandem method of DGUC followed by FPLC, which also produces high quality HDL samples for down-stream RNA isolation and sRNA quantification. Due to recent advances in high-throughput sequencing of sRNAs (sRNAseq), e.g., miRNAs, and the increased awareness of other non-miRNA sRNA classes, sRNAseq is the current state-of-the-art in miRNA and sRNA profiling. As such, our protocol recommends quantifying miRNAs and other sRNAs on HDL samples using sRNAseq. Nonetheless, total RNA isolated from HDL can also be used to quantify individual miRNAs and other sRNAs or validate sRNAseq results using real-time PCR approaches. Here we describe in detail a protocol for the collection, purification, quantification, data analysis, and validation of highly pure HDL-sRNAs.

The overall goal of this paper is to demonstrate the feasibility and process of sRNA quantification in highly pure HDL isolated from human plasma.

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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 overlay solution #1 over the plasma (Table 1).
      NOTE: The bent needle ensures all the VLDL/IDL, LDL, and HDL are removed without mixing with the overlay.
    4. Remove the majority of the bubbles at the top, leaving 2 - 3 mm space from meniscus to the top of the tube and carefully place the tubes in SW-40Ti buckets.
    5. Weigh each bucket (with the caps) on the balance, and exactly balance with the opposite bucket (bucket + cap): #1 to #4, #2 to #5, and #3 to #6.
      NOTE: These buckets must be balanced and matched at all times.
    6. Place rotor in ultracentrifuge (List of Materials). Centrifuge at 274,400 x g for 24 h at 4 °C with a #4 intermediate brake.
    7. Carefully remove buckets from rotor and the tubes from buckets.
    8. Carefully remove 2 mL from the top layer of the overlay using a syringe and bent needle. This will be the VLDL/IDL fraction which can be stored at 4 °C or -80 °C.
    9. After removing the VLDL/IDL fraction, collect the remaining 7 mL of sample (plasma) and place it into a 15 mL conical tube. Bring the volume of the sample up to 9 mL with overlay solution #2 (Table 1).
    10. Adjust the sample density from 1.025 g/mL to 1.080 g/mL with KBr using approximately 0.746 g KBr in the 9 mL sample or 0.0828 g/mL KBr of sample. Gently rock the sample until all the KBr is dissolved (approximately 20 min) and transfer to ultracentrifuge tube while ensuring bubbles rise to the top.
    11. With syringe and needle, carefully place 3 mL of overlay solution #3 over the sample (Table 1). Leave 2 - 3 mm space from meniscus to the top of the tube.
    12. Remove the majority of any remaining bubbles at the top of the tube and carefully place the filled ultracentrifuge tubes in SW-40Ti buckets.
    13. Weigh each bucket (with the caps) on the balance, and exactly balance with the opposite bucket + cap, as in 1.1.5.
    14. Place rotor in ultracentrifuge (see List of Materials). Centrifuge at 274,400 x g for 24 h at 4 °C with a #4 intermediate brake.
    15. Carefully remove buckets from rotor and the tubes from buckets.
    16. Carefully remove 2 mL from the top layer of the overlay using a syringe and bent needle. This will be the LDL fraction which can be stored at 4 °C or -80 °C.
    17. After removing the LDL fraction, collect the remaining approximate 7 mL of sample (plasma) and place it into a new 15 mL conical tube. Bring the volume of the sample (plasma) up to 9 mL with 1.080 g/mL solution #4 (Table 1).
    18. Adjust the sample density from 1.080 g/mL to 1.30 g/mL with KBr using 3.33 g KBr in the 9 mL sample (plasma) or 0.37 g KBr for each mL of sample. Rock or slightly agitate the sample until all the KBr (salt) is dissolved and transfer to ultracentrifuge tube.
    19. With syringe and needle, carefully place 3 mL of overlay solution #5 over the sample (Table 1).
    20. Remove the majority of any remaining bubbles at the top of the tube, leaving 2- 3 mm space from meniscus to the top of the tube and carefully place the filled ultracentrifuge tubes in SW-40Ti buckets.
    21. Weigh each bucket (with the caps) on the balance, and exactly balance with the opposite bucket + cap, as in 1.1.5.
    22. Place rotor in ultracentrifuge (see List of Materials). Centrifuge at 274,400 x g for 48 h at 4 °C with a #4 brake.
    23. Carefully remove buckets from rotor and the tubes from buckets.
    24. Carefully remove approximately 2 mL from the top layer of the overlay using a syringe and bent needle. This is the HDL fraction, which can be stored at 4 °C or -80 °C.
      NOTE: Following DGUC HDL may be dialyzed to remove the high-salt solutions.
    25. To dialyze, place the collected HDL fraction in a sealed dialysis sleeve (10,000 m.w. cut-off) and dialyze overnight in 1 L 1x PBS. Change dialysis buffer (1x PBS) 3 times over 24 h. Gentle perturbation of the PBS with a magnetic stir-bar will improve dialysis.
    26. Determine the protein concentration of the DGUC-HDL using a BCA method8.
  2. Fast-Protein Liquid Chromatography (FPLC) or Size-Exclusion Chromatography (~ 6 h)
    1. Filter 1.2 mg of DGUC-HDL (total protein) in 500 µL through a 0.22 µm micro-centrifugal filter (see List of Materials), immediately prior to injection.
      NOTE: An additional filtering of the DGUC-HDL sample through a 0.45 µm micro-centrifugal filter prior to the 0.22 µm filter may be required for some samples.
    2. Collect the filtered DGUC-HDL sample, load the FPLC (fill) injection syringe, and ensure there are no bubbles in the syringe before injecting into the FPLC.
    3. Set the FPLC flow rate to 0.3 mL/min with a pressure limit at 2.6 MPa. Equilibrate columns with 0.2 column volumes (approximately 15 mL) of buffer, prior to sample injection. Inject the sample with 3 mL of buffer into the FPLC (injection loop) instrument and start the run. Collect 1.5 mL fractions for a total of 72 fractions.

2. High-throughput Small RNA Sequencing (sRNAseq, ~ 9 days)

  1. RNA Isolation (~ 1 day)
    1. Identify the FPLC fractions corresponding to DGUC-HDL by determining total cholesterol levels using a colorimetric kit according to the manufacturer's instructions. Using this FPLC set-up, expect to find 6 - 7 FPLC fractions containing DGUC-HDL.
    2. Collect all the volume (approximately 8 -1 0 mL pool of 1.45 mL fraction volumes) from DGUC-HDL FPLC fractions and concentrate using 10 kDa m.w. cut-off centrifugal filter units at 4,000 x g for 1 h at 4 °C or until HDL concentrate is approximately 100 µL.
    3. Collect the HDL concentrate and quantify HDL total protein concentration using the BCA method8.
    4. Determine the highest concentration of HDL total protein, up to 1 mg, that can be aliquoted from each sample in the set. For example, isolate RNA from the same amount of HDL total protein for each sample.
    5. Perform RNA Isolation using a modified protocol (see List of Materials).
      1. Add 10x volume of phenol lysis reagent (see List of Materials) sample and vortex for 1 min.
      2. Incubate at room temperature for 5 min.
      3. Add chloroform (20% of phenol lysis reagent volume used in step 2.1.5.1) and shake vigorously for 15 s.
      4. Incubate at room temperature for 2 - 3 min.
      5. Centrifuge for 15 min at 12,000 x g at 4 °C in a refrigerated centrifuge.
      6. Transfer the upper aqueous phase to a new tube, avoiding the interphase.
      7. Add 1.5x volume of 100% ethanol to the aqueous phase and vortex for 1 min.
      8. Store samples for at least 1 h or overnight at -80 °C.
      9. Continue with the RNA isolation protocol using the provided mini columns.
      10. Elute with 30 µL of RNase-free H2O. First add 15 µL of H2O directly to the frit, spin at low speed (2,000 x g) for 2 min, and then spin at high speed (max) for 1 min. Repeat this step with an additional 15 µL of H2O.
    6. Immediately proceed to sRNA library generation or store at -80 °C.
  2. Library Generation (~ 6 h)
    1. Prepare sRNA cDNA libraries using the sRNA library generation kit protocol, as per manufacturer's instructions with one modification to the PCR amplification as detailed below.
      1. Prepare the PCR mastermix on ice containing 0.5 µL DNase/RNase-free H2O, 15 µL PCR Mix (PML) and 1 µL RNA PCR Primer (RP1) per sample (include an additional 10% for pipetting error).
      2. Add 16.5 µL of the PCR mix to each (cDNA) sample.
      3. Assign an index/barcode number to each sample for multiplexing and add 1 µL PCR Primer Index (to the corresponding number) to the sample.
      4. Perform a quick spin using the microfuge.
        NOTE: Total volume should now be 30 µL per sample.
      5. Perform the cycling conditions for PCR amplification as detailed in Tables 2 and 3.
  3. Size Select sRNA Library to Remove Adapter:Adapters (~ 1.5 h)
    1. Perform library size-selection using automatic DNA size selector as per manufacturer's instructions with the following size parameters: Target: 156 bp, Start: 135 bp, End: 177 bp, Range Flag: Broad.
  4. DNA Clean Up (~0.5 h)
    1. Clean up size-selected sRNA cDNA according to the manufacturer's instructions.
    2. Elute clean and concentrated sRNA cDNA with 2 x 7 µL DNase/RNase-free H2O.
  5. Quantify and Assess sRNA cDNA Library Yield and Quality (~ 1 h)
    1. Assess sRNA cDNA library quality and size using a high-sensitivity DNA chip on a bioanalyzer (List of Materials) as per manufacturer's instructions.
    2. Quantify the individual sRNA cDNA library concentrations using a high-sensitivity DNA assay (List of Materials), as per manufacturer's instructions.
      NOTE: It is recommended to have all samples with different indices be run on the same lane of the flow cell if possible. If more than one lane is required, mix case and control samples appropriately.
  6. High-throughput sRNA Sequencing (~ 7 days)
    1. Pool individual indexed sRNA libraries based on equimolar concentrations.
    2. Screen pooled sRNA libraries for quality using high-sensitivity DNA chip on the bioanalyzer and determine library DNA concentration as in 2.5.1 - 2.5.2.
    3. Perform quantitative PCR (qPCR) of adapter content to ensure appropriate sequencing depth using the sequencing library qPCR quantification kit as per manufacturer's instructions (List of Materials).
    4. Perform sequencing using a single read, 50 bp protocol to reach a depth of approximately 20 - 25 M reads/sample, as per manufacturer's instructions.

3. Data Analysis (~ 1 day)

  1. Use bcl2fastq2, as per user guide instructions, to preprocess raw fastq files of demultiplexed samples (List of Materials).
  2. Use Cutadapt to trim adapters9 and remove reads < 16 nucleotides (nts) in length, as per user guide instructions (List of Materials).
  3. Remove contamination sequences present in the sRNA libraries, including stop solution sequence (STP: CCACGTTCCCGTGG) and adapter carry-over (CTACAGTCCGACGATC) using removeSequenceInFastq function in NGSPERL framework, as per user guide instructions (List of Materials).
  4. Perform quality control metrics by FastQC using Center for Quantitative Sciences (CQS) tools, as per user guide instructions (List of Materials).
  5. Generate non-redundant list of "identical" reads (collapse redundant reads) and record copy numbers using CQSTools, as per user guide instructions (List of Materials).
  6. Align reads to human genome using Bowtie1.1.2 allowing 1 mismatch (options: -a -m 100 --best --strata -v 1), as per user guide instructions (List of Materials).
  7. Perform read alignment, counting, and result summarization tasks using NGSPERL, as per user guide instructions.
  8. Convert exported count table to a spreadsheet file.
  9. Normalize reads of a specific class (e.g., miRNAs) to total number of reads for that class (e.g., total number of miRNAs reads) and report signals as Reads Per Mapped miRNA (RPMM). (e.g., RPMM =(miR-X/Total # of miRNA reads)*1,000,000).
  10. Input normalized count table as generic single color technology into software for low-level and high-level differential analyses as per user guide instructions (List of Materials).
    NOTE: Currently, there are no well-characterized housekeeping genes/sRNAs for HDL-sRNA. A spike-in control may be used, but can be problematic. Normalizing to HDL total protein input or volume is advised. Most importantly, it is recommended to validate sequencing results by one of the various strategies to quantify miRNAs and sRNAs using real-time PCR or quantitative PCR.

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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...

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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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by awards from the National Institutes of Health, National Heart, Lung and Blood Institute to K.C.V. HL128996, HL113039, and HL116263. This work was also supported by awards from the American Heart Association to K.C.V. CSA2066001, D.L.M POST26630003, and R.M.A. POST25710170.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Ultracentrifuge Beckman CoulterA99839Optima XPN-80
Ultracentrifuge RotorBeckman Coulter331362SW-41Ti
AKTA Pure FPLC SystemGE Healthcare29018224
3x FPLC Superdex 200 Increase Columns In-lineGE Healthcare2899094410/300 gl
SynergyMxBioTek Instruments7191000
Tabletop centrifugeThermo Scientific75004525Sorvall ST40R
Refrigerated centrifugeEppendorf226298675417R (purchased through USA Scientific)
Microfuge USA Scientific2631-0006
PippenPrepSage SciencePIP0001
2100 Bioanalyzer AgilentG2938B
High Sensitivity DNA AssayAgilent5067-4626
Sequencing Library qPCR Quantification KitIlluminaSY-930-1010
ProFlex Thermal CyclerApplied Biosystems4484073
QuantStudio 12k FlexApplied Biosystems4471134
EpMotion RobotEppendorf9600001115070
Ultra-clear centrifuge tubesBeckman Coulter344059
Potassium BromideFisher ChemicalsP205-500
15 mL conical tubeThermo Scientific339650
Micro-centrifugal filters 0.45 µmMilliporeUFC30HV00
Micro-centrifugal filters 0.22 µmMilliporeUFC30GV00
miRNAEasy Total RNA Isolation KitsQiagen217004
Total Cholesterol colormetric kitCliniqa (Raichem)R80035
10,000 m.w. cut-off centrifugation filterAmiconUFC801024purchased through Millipore
PCR strip tubesAxygenPCR-0208-Cpurchased through Fisher
microRNA RT kitLife Technologies4366597For 1000 reactions
PCR master mixLife Technologies444004150 mL bottle
Pierce BCA kitThermo Scientific23225
Clean and Concentrator KitZymoD4014
Dialysis tubingSpectrum Labs132118purchased through Fisher
bcl2fastq2Illuminan/aSoftware
Cutadapthttps://github.com/marcelm/cutadaptn/aSoftware
NGSPERLgithub.com/shengqh/ngsperln/aSoftware
CQSToolsgithub.com/shengqh/CQS.Toolsn/aSoftware
Bowtie 1.1.2 http://bowtie-bio.sourceforge.netn/aSoftware
GeneSpringGX13.1.1Agilentn/aSoftware

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Small Non coding RNADensity gradient UltracentrifugationFast protein Liquid ChromatographyHDL IsolationLipoprotein ProfilingPlasma FractionationCholesterol MeasurementBCA Protein Assay
Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification
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