Exosomal RNA isolation from urine samples

The Urine Exosome RNA Isolation Kit extracts exosomal RNA from urine samples quickly and efficiently.

Image Credit: Norgen Biotek Corp.

Image Credit: Norgen Biotek Corp.

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Format

Slurry

Size

50 preps

Overview

The regimen can be performed in under 50 minutes. Urine quantities ranging from 1 to 10 mL can be processed conveniently and quickly. All sizes of RNA are recovered at the same rate, eliminating the need for harmful chemicals such as phenol.

Features and benefits

• Isolating exosomal RNA molecules from urine samples
• Quick and convenient spin-column process
• For most downstream uses, such as small RNA sequencing, isolate inhibitor-free urine microRNA. Find out more about the NGS services offered by Norgen
• Optional procedure for exosomal protein purification in preparation for western blot analysis

Background

Most cell types secrete exosomes, membrane vesicles ranging in size from 40 to 150 nm. Among other biological fluids, exosomes are present in saliva, blood, urine, amniotic fluid, and malignant ascite fluids. These vesicles communicate with distant cells and tissues throughout the body by acting as cellular messengers. Cell-specific proteins, lipids, and RNAs found in exosomes are transferred to other cells where they can alter physiology and/or function.

These exosomes might be involved in tissue repair, neural communication, pathogenic protein transfer, and adaptive immune responses to infections and tumors. According to recent research, exosomes contain unique subsets of microRNAs that can reveal information about the type of cell that secretes them.

Exosomal RNAs may, therefore, be used as biomarkers for several illnesses, including cancer. Since RNases prevent the RNA molecules enclosed in exosomes from degradation, they can be effectively extracted from bodily fluids like urine.

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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. Image Credit: Norgen Biotek Corp.

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. Image Credit: Norgen Biotek Corp.

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. Image Credit: Norgen Biotek Corp.

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. Image Credit: Norgen Biotek Corp. Image Credit: Norgen Biotek Corp.

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. Image Credit: Norgen Biotek Corp.

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.). Image Credit: Norgen Biotek Corp.

Figure 7. EV pellets recovered by Norgen’s Urine Exosome RNA Isolation Kit (Cat. 47200) kit were visualized 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 normalization for serum and urine. J Immunol Methods. 2016 Feb;429:39-49.). Image Credit: Norgen Biotek Corp.

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Kit specifications

Source: Norgen Biotek Corp.

Storage conditions and product stability

All buffers should be properly sealed and stored at room temperature. This kit is stable for two years from the date of dispatch.

Source: Norgen Biotek Corp.