Protocols

   

The consortium has developed Standard Operating Procedures (SOPs) for purification of extracellular vesicles and RNA, as well as for exRNA sequencing and data analysis. A major goal of ERCC Stage 1 was to standardize these procedures to minimize experimental variation across labs and experiments.

Most of the protocols were updated after first release in 2015 based on vetting the originals in multiple labs. The major change to isolation and enrichment protocols was an increase in input volume of biofluid from 200 µL to 500 µL. The larger input volume yields more reproducible amounts of RNA isolation across multiple experiments.

Updated protocols are indicated here as version 2 (v2). We have maintained links to version 1 protocols for archival purposes.

   

miRDaR

miRDaR is a web application designed to help researchers select the best RNA isolation protocol for detection and reproducibility of miRNAs of interest to them. It accompanies the publication Srinivasan et al. Small RNA sequencing across diverse biofluids identifies optimal methods for exRNA isolation. Cell (2019) and replaces the earlier protocols decision tree, still linked here for archival purposes.

 

Protocols Decision Tree

This decision tree allows a beginner to understand the best approaches available in the field to study extracellular vesicles and extracellular RNA. See this blog post for an in-depth discussion of how to select protocols from this page. The Protocols Decision Tree currently still refers to version 1 protocols.

 

Collection of Biofluids

• Collection of Cell Culture Supernatant
• Bile Sample Collection for the analysis of extracellular RNA
• Plasma Collection Procedure (Small Scale) for the analysis of extracellular RNA
• Serum Collection Procedure (Small Scale) for the analysis of extracellular RNA
• Urine Collection Procedure for the analysis of extracellular RNA

 

Isolation and Enrichment of Extracellular Vesicles

• Exosome isolation from plasma using ExoQuick reagent (v2)
• (Version 1)
• Exosome isolation from serum using ExoQuick reagent (v2)
• (Version 1)
• Enrichment of extracellular vesicles using Millipore membrane filters followed by isolation of exosomal RNA from serum or plasma using the Qiagen miRNeasy Micro kit (v2)
• Version 1: Enrichment of EVs using Millipore membrane filters
• Isolation of exosomal RNA from serum or plasma using ultracentrifugation and the Qiagen miRNeasy Micro kit (v2)
• Version 1: Enrichment of EVs from serum or plasma via ultracentrifugation
• Improved exosome isolation by sucrose gradient fractionation of ultracentrifuged crude exosome pellets

 

RNA Isolation

• Small RNA sequencing across diverse biofluids identifies optimal methods for exRNA isolation  
• Human plasma and serum extracellular small RNA reference profiles and their clinical utility

  All of the protocols below can be found in the Supplementary Appendix of the publication above.

  • Total RNA isolation from urine, serum, citrate-, EDTA–, and heparin–plasma samples
  • Full protocol for barcoded cDNA library preparation for small RNA sequencing
  • Expression, purification, and quality control of RNA ligases
 
• Isolation of extracellular vesicle RNA from serum
• Isolation of extracellular vesicle RNA from bile
• Isolation of extracellular vesicle RNA from cell culture supernatant
• Isolation of exosomal RNA from serum or plasma using the Qiagen ExoRNeasy Midi kit (v2)
• Version 1: RNA isolation using the ExoRNeasy Mini Kit
• Isolation of exosomal RNA from urine using the Qiagen miRNeasy Micro kit (v2)
• Version 1: RNA isolation using the miRNeasy Micro Kit
• Isolation of exosomal RNA from serum or plasma using the New England Peptide ME^TM kit (v2)
• (Version 1)
• Isolation of exosomal RNA from serum or plasma using the miRcury Biofluids kit (v2)
• Version 1: RNA isolation using the miRcury Biofluids Kit
• Isolation of exosomal RNA from serum or plasma using the Qiagen miRNeasy Micro kit (v2)
• Version 1: RNA isolation using the miRNeasy Micro Kit
• Isolation of exosomal RNA from serum or plasma using the Norgen BioTek Plasma/Serum circulating and exosomal RNA purification mini kit (v2)
• Version 1: RNA Isolation from human serum and plasma using the Norgen Kit
• Extracellular RNA isolation using the SeraMir Kit (v1)

 

Validation and Quantification

• qPCR of Genomic DNA to assess DNA contamination of extracellular RNA samples
• qRT-PCR
• RiboGreen Quantification via qPCR machine
• Quality Control of Serum and Plasma

 

RNA Sequencing

• Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling

  This publication includes four variants on the main idea of adding random adapters at the amplification stage of sequencing.

  • In house 4N Protocol A: Library Preparation for small RNA sequencing using 4N adapters
  • In house 4N Protocol B: Library Preparation for small RNA sequencing using 4N adapters
  • In house 4N Protocol C: Library Preparation for small RNA sequencing using 4N adapters
  • In house 4N Protocol D: Modified TruSeq Small RNA Library Prep using Randomized 4N Adapters
  • An early draft of the protocol is still available here.
 
• Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing

This protocol is an early version of the one outlined in Max et al (PNAS 2018). That publication is here, and the protocol itself is in its Supplementary Appendix.

• RNA Sequencing Analysis of Salivary Extracellular RNA

 

Proteomics on Extracellular Vesicles

• Proteomic Characterization of Exosomes from HIV-1-Infected Cells