RNA Quantification Kit for Purified EV
Extracellular vesicles (EVs), including exosomes, contain various RNAs such as mRNA and miRNA. These RNAs are considered key functional molecules in intercellular communication, leading to active research in various fields including diagnostics and therapeutics. Next-generation sequencing (NGS) and microarrays, used for comprehensive analysis of RNA within EVs, require prior measurement of RNA quantities.
Fujifilm Wako offers an RNA quantification kit capable of measuring even trace amounts of RNA present in purified EVs.
Product Overview
The RNA Quantification Kit for Purified EV is designed to measure total RNA extracted from extracellular vesicle (EV) solutions, including exosomes. The kit uses an RNA-binding fluorescent dye to enable highly sensitive detection of trace amounts of RNA in solution. It is ideal for RNA quantification prior to next-generation sequencing (NGS) or microarray analysis in EV research.
Features
- Suitable for small-volume RNA samples (1-20 μL)
- High sensitivity: 0.1-10 ng/μL
- Rapid assay: 5-minute reaction time
Sample
Purified EV
For isolating and purifying EVs, the MagCapture™ Exosome Isolation Kit PS Ver.2 (Product No. 290-84103) is recommended. For extracting and purifying RNA, the ｍicroRNA Extractor™ Kit for Purified EV (Product No. 294-84601) is recommended.
Protocol
1. Reagent Preparation
- 1x Dilution Buffer
- : Add nuclease-free water to Dilution Buffer (20x) at a ratio of 1 : 20 and mix well.
- Fluorescent Buffer
- : Add 1x Dilution Buffer to the Fluorescent Reagent at a ratio of 1:2,000 and mix well.
(Protect from light by wrapping in aluminum foil, etc.) - Control RNA Solution
- : Dilute the Control RNA (100 ng/μL) stepwise as shown below to prepare Control RNA Solutions of each concentration.
| Concentration of the Control RNA Solution (ng/μL) |
Volume of the Control RNA Solutions |
Volume of 1x Dilution Buffer |
|---|---|---|
| 100 | Stock Control RNA: 100 μL | - |
| 10 | 100 ng/μL solution: 10 μL | 90 μL |
| 5 | 10 ng/μL solution: 50 μL | 50 μL |
| 2.5 | 5 ng/μL solution: 50 μL | 50 μL |
| 1.25 | 2.5 ng/μL solution: 50 μL | 50 μL |
| 0.625 | 1.25 ng/μL solution: 50 μL | 50 μL |
| 0.3125 | 0.625 ng/μL solution: 50 μL | 50 μL |
| 0.15625 | 0.3125 ng/μL solution: 50 μL | 50 μL |
The table above shows one example of dilution; adjust the concentration of the control RNA solution according to your sample.
For example, the following serial dilution is recommended when quantifying RNA extracted from EV derived from COLO201 cells.
| Concentration of the Control RNA Solution (ng/μL) |
Volume of the Control RNA Solutions |
Volume of 1x Dilution Buffer |
|---|---|---|
| 100 | Stock Control RNA: 100 μL | - |
| 1 | 100 ng/μL solution: 2 μL | 198 μL |
| 0.5 | 10 ng/μL solution: 50 μL | 50 μL |
| 0.25 | 5 ng/μL solution: 50 μL | 50 μL |
| 0.125 | 2.5 ng/μL solution: 50 μL | 50 μL |
| 0.0625 | 1.25 ng/μL solution: 50 μL | 50 μL |
| 0.03125 | 0.625 ng/μL solution: 50 μL | 50 μL |
| 0.015625 | 0.3125 ng/μL solution: 50 μL | 50 μL |
2. RNA Quantification
- Dispense 200 μL of Fluorescent Buffer into each well.
- Dispense 10 μL of Control RNA Solution into the wells designated for standards.Add 10 μL of 1x Dilution Buffer to the blank.
- Dispense 1-20 μL of pretreated-RNA samples into the wells designated for samples.
- Agitate the mixture in each well using a microplate shaker or by pipetting.
- Incubate at room temperature for 5 minutes in the dark.
- Determine the fluorescent intensity using the 96-well microplate reader(excitation: 492 nm; emission: 540 nm).
Example of plate layout (quantifying RNA extracted from EV derived from COLO201 cells)
Data
Quantification of RNA from Purified EVs
EVs were isolated from umbilical cord-derived mesenchymal stem cells using the MagCapture™ Exosome Isolation Kit PS Ver.2 (Product No. 290-84103), and total RNA was extracted using the microRNA Extractor™ Kit for Purified EVs (Product No. 294-84601). A 5 μL aliquot of the extracted RNA solution was then quantified with this kit. The results confirmed that RNA derived from EVs can be reliably quantified using this product.
(1) Calibration Curve
(2) Quantification of RNA
| Measured value (RFU ⊿Blank) | RNA amount (ng) |
|---|---|
| 4,239 | 0.977 |
[Result]
This product can be used to quantify RNA in EVs.
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For research use or further manufacturing use only. Not for use in diagnostic procedures.
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