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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

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

Published: November 28th, 2016

DOI:

10.3791/54488

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

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

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.

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....

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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 .......

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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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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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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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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 0.45µm Millipore UFC30HV00
Micro-centrifugal filters 0.22µm 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

  1. Vickers, K. C., Roteta, L. A., Hucheson-Dilks, H., Han, L., Guo, Y. Mining diverse small RNA species in the deep transcriptome. Trends Biochem Sci. 40, 4-7 (2015).
  2. Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116, 281-297 (2004).
  3. Haussecker, D., et al. Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA. 16, 673-695 (2010).
  4. Li, Z., et al. Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs. Nucleic Acids Res. 40, 6787-6799 (2012).
  5. Martens-Uzunova, E. S., Olvedy, M., Jenster, G. Beyond microRNA--novel RNAs derived from small non-coding RNA and their implication in cancer. Cancer Lett. 340, 201-211 (2013).
  6. Maute, R. L., et al. tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma. Proc Natl Acad Sci U S A. 110, 1404-1409 (2013).
  7. Vickers, K. C., Palmisano, B. T., Shoucri, B. M., Shamburek, R. D., Remaley, A. T. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 13, 423-433 (2011).
  8. Smith, P. K., et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 150, 76-85 (1985).
  9. Chen, C., Khaleel, S. S., Huang, H., Wu, C. H. Software for pre-processing Illumina next-generation sequencing short read sequences. Source Code Biol Med. 9, 8 (2014).
  10. Vickers, K. C., Remaley, A. T. Lipid-based carriers of microRNAs and intercellular communication. Curr Opin Lipidol. 23, 91-97 (2012).
  11. Greening, D. W., Xu, R., Ji, H., Tauro, B. J., Simpson, R. J. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. Methods Mol Biol. 1295, 179-209 (2015).
  12. Sung, B. H., Ketova, T., Hoshino, D., Zijlstra, A., Weaver, A. M. Directional cell movement through tissues is controlled by exosome secretion. Nat Commun. 6, 7164 (2015).
  13. Livak, K. J., Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25, 402-408 (2001).
  14. Jung, R., Lubcke, C., Wagener, C., Neumaier, M. Reversal of RT-PCR inhibition observed in heparinized clinical specimens. Biotechniques. 23, (1997).
  15. Johnson, M. L., Navanukraw, C., Grazul-Bilska, A. T., Reynolds, L. P., Redmer, D. A. Heparinase treatment of RNA before quantitative real-time RT-PCR. Biotechniques. 35, 1140-1142 (2003).
  16. Garcia, M. E., Blanco, J. L., Caballero, J., Gargallo-Viola, D. Anticoagulants interfere with PCR used to diagnose invasive aspergillosis. J Clin Microbiol. 40, 1567-1568 (2002).
  17. Yokota, M., Tatsumi, N., Nathalang, O., Yamada, T., Tsuda, I. Effects of heparin on polymerase chain reaction for blood white cells. J Clin Lab Anal. 13, 133-140 (1999).
  18. Bai, X., et al. Predictive value of quantitative PCR-based viral burden analysis for eight human herpesviruses in pediatric solid organ transplant patients. J Mol Diagn. 2, 191-201 (2000).
  19. McShine, R. L., Sibinga, S., Brozovic, B. Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean platelet volume. Clin Lab Haematol. 12, 277-285 (1990).
  20. Fichtlscherer, S., et al. Circulating microRNAs in patients with coronary artery disease. Circ Res. 107, 677-684 (2010).
  21. Pritchard, C. C., Cheng, H. H., Tewari, M. MicroRNA profiling: approaches and considerations. Nat Rev Genet. 13, 358-369 (2012).
  22. Kirschner, M. B., et al. Haemolysis during sample preparation alters microRNA content of plasma. PLoS One. 6, e24145 (2011).
  23. Kannan, M., Atreya, C. Differential profiling of human red blood cells during storage for 52 selected microRNAs. Transfusion. 50, 1581-1588 (2010).
  24. Chen, S. Y., Wang, Y., Telen, M. J., Chi, J. T. The genomic analysis of erythrocyte microRNA expression in sickle cell diseases. PLoS One. 3, e2360 (2008).
  25. Balzano, F., et al. miRNA Stability in Frozen Plasma Samples. Molecules. 20, 19030-19040 (2015).
  26. Redgrave, T. G., Roberts, D. C., West, C. E. Separation of plasma lipoproteins by density-gradient ultracentrifugation. Anal Biochem. 65, 42-49 (1975).
  27. Cheung, M. C., Wolf, A. C. Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations. J Lipid Res. 29, 15-25 (1988).
  28. Kunitake, S. T., Kane, J. P. Factors affecting the integrity of high density lipoproteins in the ultracentrifuge. J Lipid Res. 23, 936-940 (1982).
  29. Laurent, L. C., et al. Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium. J Extracell Vesicles. 4, 26533 (2015).

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