Name: Urine Exosome RNA Isolation Kit
Brand: Norgen Biotek Corp.
SKU: 47200
Price: 715.000000 USD
Availability: InStock

Features and Benefits

Isolation of exosomal RNA molecules from urine samples
Rapid and convenient spin-column protocol
Isolate inhibitor-free urinary microRNA for most downstream applications, including Small RNA Sequencing. Find out more information on Norgen's NGS services
Optional protocol for purification of exosomal proteins for western blot analysis

This kit provides a rapid spin column procedure for the isolation of exosomal RNA from urine samples. Users can simultaneously concentrate and isolate high quality exosomal RNA, including microRNA, for use in sensitive downstream assays such as RT-PCR, qRT-PCR, NGS, microarrays and more. The protocol can be completed in under 50 minutes. Urine volumes of 1 to 10 mL can be processed easily and rapidly. All sizes of RNA are recovered at an equal rate without the need for using hazardous chemicals like phenol.

Background

Exosomes are 40 - 150 nm membrane vesicles, which are secreted by most cell types. Exosomes can be found in saliva, blood, urine, amniotic fluid and malignant ascite fluids, among other biological fluids. These vesicles act as cellular messengers, conveying information to distant cells and tissues within the body. The exosomes contain cell-specific proteins, lipids and RNAs, which are transported to other cells, where they can alter function and/or physiology. These exosomes may play a functional role in mediating adaptive immune responses to infectious agents and tumours, tissue repair, neural communication and transfer of pathogenic proteins. Recent work has demonstrated the presence of distinct subsets of microRNAs within exosomes which can inform about the cell type from which the exosomes are secreted. For this reason, exosomal RNAs may serve as biomarkers for various diseases including cancer. As the RNA molecules encapsulated within exosomes are protected from degradation by RNases they can be efficiently recovered from biological fluids, such as urine.

Details

Supporting Data

Figure 1. Detection of Urine Exosomal RNA Isolated from Different Urine Volumes. Norgen's Urine Exosome RNA Isolation Kit was used to isolate urine exosomal RNA from different urine volumes ranging from 1 to 15 mL. The purified urine exosomal RNA was then used as the template in an RT-qPCR reaction to detect the human 5S rRNA. 3 μL of the isolated urine exosomal RNA was used as the template in the RT step, and 3 µL from the RT step was used in the qPCR reaction. As it can be seen, the qPCR was able to successfully detect and amplify the 5S rRNA from exosomal RNA isolated from different urine volumes, indicating its high quality.

Figure 2. Effective and Consistent Detection of Urine Exosome RNA. Norgen's Urine Exosome RNA Isolation Kit can effectively isolate RNA from urine. Urine exosome RNA was isolated from 5 mL of human cell-free urine using Norgen's Urine Exosome RNA Isolation Kit (green), ExoQuick-TC Exosome Precipitation Reagent (blue) and Amicon® Ultra-15 Filter (blue). Stem loop RT-qPCR using primers specific to miR-21 and miR-16 as well as the housekeeping 5S rRNA was performed. In brief, three microliters of the 100 µL isolated RNA was then subjected to a 20 µL reverse transcription reaction using 5S rRNA, miR-21 and miR-16 stem-loop reverse primer or reverse primer. 3 μL from the reverse transcription reaction was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21, human miR-16 and the 5S rRNA. Norgen's Urine Exosome RNA Isolation Kit is the only product that showed consistent detection of all tested transcripts with the highest quality.

Figure 3. High Diversity and Consistency of miRNA Recovered from Urine. Mid-stream urine was collected from three different healthy individuals. RNA was isolated from 20 mL of each sample using Norgen's Urine Exosome RNA Purification Kit (Cat. 47200). Small RNA Libraries were then generated using an Illumina TruSeq Small RNA Library Preparation Kit and subsequently sequenced on an Illumina MiSeq system. The normalized read counts of the mapped miRNAs were then compared between each individual sample. The scatter plots showed that the miRNA diversity was highly conserved among all individuals tested (R² ≥ 0.92). This suggests that Norgen's RNA purification workflow recovers consistent profiles of small RNAs. This could enhance the possibility of detecting potential urine-based small RNA biomarkers.

Figure 4. High Degree of Overlapping of miRNA profile between Urine and Plasma. Plasma and mid-stream urine was collected from three different healthy individuals. RNA was isolated from 20 mL of each urine sample using Norgen's Urine Exosomal RNA Purification Kit (Cat. 47200) and 200 µL of each plasma sample using Norgen's Plasma/Serum RNA Purification Mini Kit (Cat. 55000). Small RNA Libraries were then generated using an Illumina TruSeq Small RNA Library Preparation Kit and subsequently sequenced on an Illumina MiSeq system. The list of mapped miRNAs from plasma and urine from the same individual was then compared. The Venn diagrams showed that the miRNA profiles have a high degree of overlapping between urine and plasma within each individual.

Figure 5. Increased Diversity of Other Small RNA Species (Piwi-Interacting) in Urine. Plasma and mid-stream urine was collected from three different healthy individuals. RNA was isolated from 20 mL of each urine sample using Norgen's Urine Exosomal RNA Purification Kit (Cat. 47200) and 200 µL of each plasma sample using Norgen's Plasma/Serum RNA Purification Mini Kit (Cat. 55000). Small RNA Libraries were then generated using an Illumina TruSeq Small RNA Library Preparation Kit and subsequently sequenced on an Illumina MiSeq system. The above chart shows that the relative proportion of piwi-interacting RNA (piRNA) was consistently higher in urine.

Figure 6. Western blot analysis of Norgen-enriched uEVs (urinary Extracellular Vesicles) from BPH (benign prostatic hyperplasia) and PCa (Pancreatic Cancer) urine samples. (Royo F, et al. Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples. Journal of Extracellular Vesicles. 2016;5:10.)

Figure 7. EV pellets recovered by Norgen’s Urine Exosome RNA Isolation Kit (Cat. 47200) kit were visualised by EM (Electron Microscopy) for sizing (Crossland RE, Norden J, Bibby LA, Davis J, Dickinson AM. Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine. J Immunol Methods. 2016 Feb;429:39-49.)

Kit Specifications
Minimum Urine Input	1 mL
Maximum Urine Input	10 mL
Size of RNA Purified	Small exosomal RNA species
Time to Complete Purification	~ 50 minutes

Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.

Component	Cat. 47200 (50 preps)
Slurry B1	18 mL
Binding Buffer A	20 mL
Lysis Buffer A	2 x 20 mL
Wash Solution A	38 mL
Elution Solution A	6 mL
Mini Filter Spin Columns	50
Collection Tubes	50
Elution Tubes (1.7 mL)	50
Product Insert	1

Documentation

PROTOCOLS

(47200) Urine Exosome RNA Isolation Kit - Protocol (50 prep)

FLYERS

Urine Exosome RNA Isolation Kit

FAQs

Spin Column

What should I do if some of the grey resin is transferred out of the 15 mL conical tube when I am decanting the supernatant?

Simply remix and re-centrifuge. After centrifuging decant the supernatant

What if I added more or less of the specified reagents volume?

Adding less volume may reduce RNA yields. Adding more may not affect the RNA yields except when performing the RNA elution step. Eluting RNA in higher volumes of Elution Solution A will result in diluting your RNA.

Can I perform a second elution?

Yes, you can. A second elution is possible, but it is recommended that this elution be performed in a smaller volume (50 µL).

Why do my samples show very low RNA yield?

Some biological samples will contain very little exosomal RNA. This will vary from individual to individual based on a number of variables such as how dilute the sample is before processing. In order to increase the RNA yield it may be necessary to isolate RNA from a larger volume of sample or isolate from several samples and then pool the RNA eluted from the samples.

Why does my RNA not perform well in downstream applications?

If a different elution solution was used from the one provided in the kit, the buffer should be checked for components that may interfere with the application. Common components know to interfere with downstream assays include high salts, EDTA, detergents and other denaturants. Check the compatibility of your elution buffer with its intended use.

What if my elution was contaminated with the precipitated resin?

Let the elution stand vertically for 10 minutes to precipitate the resin and then use the clear elution supernatant for downstream applications. This will neither decrease the RNA yield nor interfere with any downstream application.

How do I prepare frozen urine for exosome isolation using Norgen's Urine Purification Kits?

Urine samples stored at -80°C, -20°C or at 4°C might develop some precipitation due to the aggregation of some of the highly abundant proteins in urine. We recommend gently warming the sample to room temperature or 37°C for 5 min and to NOT perform a centrifugation step as this will eliminate the precipitated proteins leading to loss of protein-bound cf-NA or exosomes.

Can Norgen's Urine RNA isolation exosome kit be used with Animal Urine samples?

Yes, it has been used with Canine urine samples. Please contact our technical support team at support@norgenbiotek.com and ask for reference publications.

Citations

Title	Circulating and urinary microRNA profile in focal segmental glomerulosclerosis: a pilot study
Citation	Journal of Clinical Investigation 2015.
Authors	A Ramezani, JM Devaney, S Cohen, Mr Wing, R Scott, S Knoblach, R Singhal, L Howard, JB Kopp, DS Raj

Title	Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype
Citation	PNAS 2014.
Authors	JM Mc Allister, B Modi, BA Miller, J Biegler, R Bruggeman, RS Legro, JF Strauss III

Title	Emerging technologies in extracellular vesicle-based molecular diagnostics
Citation	Expert Review of Molecular Diagnostics 2014.
Authors	S Jia, D Zocco, ML Samuels, MF Chou, R Chammas, J Skog, N Zarovni, F Momen-Heravi, WP Kuo

Title	Colonic miRNA Expression/Secretion, Regulated by Intestinal Epithelial PepT1, Plays an Important Role in Cell-to-Cell Communication during Colitis
Citation	PLoS One 2014.
Authors	S Ayyandurai, MA Charania, B Xiao, E Viennois, Y Zhang, D Merlin

Title	A Simplified Method to Recover Urinary Vesicles for Clinical Applications, and Sample Banking
Citation	Scientific Reports 2014.
Authors	L Musante, D Tataruch, D Gu, A Benito-Martin, G Calzaferri, S Aherne, H Holthofer

Title	Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine
Citation	Kidney International 2013.
Authors	L Cheng, X Sun, BJ Scicluna, BM Coleman, AF Hill

Urine Exosome RNA Isolation Kit