Izon qEV RNA Extraction Kit User Manual
- June 11, 2024
- IZON
Table of Contents
q EV RNA EXTRACTION KIT
USER MANUAL
qEV RNA Extraction Kit
Izon Science Ltd. provides this document to its customers with a product
purchase to use in the product operation. This document is copyright protected
and any reproduction of the whole or any part of this document is strictly
prohibited, except with the written authorisation of Izon Science Ltd.
The contents of this document are subject to change without notice. All
technical information in this document is for reference purposes only. System
configurations and specifications in this document supersede all previous
information received by the purchaser.
Izon Science Ltd. makes no representations that this document is complete,
accurate or error-free and assumes no responsibility and will not be liable
for any errors, omissions, damage or loss that might result from any use of
this document, even if the information in the document is followed properly.
Izon products are designed and manufactured under a quality system certified
to ISO 13485:2016.
This document is not part of any sales contract between Izon Science Ltd. and
a purchaser. This document shall in no way govern or modify any Terms and
Conditions of Sale, which Terms and Conditions of Sale shall govern all
conflicting information between the two documents.
The qEV RNA Extraction Kit is produced by Norgen Biotek Corp. for Izon
Science.
Izon Science Ltd
PO Box 9292
Addington
Christchurch 8024
New Zealand
Telephone: +64 3 357 4270
Email: support@izon.com
Website: www.izon.com
DEFINITIONS AND WRITING CONVENTIONS
Make sure to follow the precautionary statements presented in this guide.
Safety and other special notices will appear in boxes and include the symbols
detailed below.
Table 1: Safety and Hazard Symbols| This symbol indicates general advice on
how to improve procedures or recommends measures to take in specific
situations
---|---
****| This symbol indicates where special care should be taken
INTRODUCTION
2.1 Intended Use
The qEV RNA Extraction Kit can be used for the extraction of RNA from EVs that
have been isolated using qEV columns, and for EVs that have been concentrated
after qEV isolation. The qEV RNA Extraction Kit enables the extraction of RNA
from EVs isolated from biofluids as well as from cell culture media. The
extraction provides samples of high purity which can be eluted into volumes of
50–100 μL (as specified by the user). The purified RNA is suitable for use in
downstream applications including qPCR, microarrays, and RNA sequencing.
MANUAL OPERATING INSTRUCTIONS
3.1 Working With RNA
RNases are very stable and robust enzymes that degrade RNA, and are not always
sufficiently removed by autoclaving solutions and glassware.
Surface contamination. All surfaces should be cleaned with commercially
available RNase decontamination solutions prior to starting experiments. RNA
work should be conducted away from microbiological workstations.
Gloves. Always wear clean, disposable gloves, and change gloves frequently.
User provided equipment. RNase-/DNase-free solutions, tips and tubes, plus
designated pipettes and lab coats should be used for all experiments involving
RNA.
RNA stability. Purified RNA should be handled and kept on ice prior to and
during downstream applications.
3.2 Kit Components
Components of the qEV RNA Extraction Kit (50 preparations)
| Components | 50 preparations |
|---|---|
| Lysis Buffer A | 2 x 30 mL |
| Lysis Additive B | 7 mL |
| Wash Solution A | 38 mL |
| Elution Solution A | 6 mL |
| Mini Spin Columns | 50 |
| Collection Tubes | 50 |
| Elution Tubes (1.7 mL) | 50 |
** Lysis Buffer A contains guanidine thiocyanate and should be handled with
care. Guanidine thiocyanate forms highly reactive compounds when combined with
bleach, thus care must be taken to properly dispose of any of this solution.
If liquid containing these buffers is spilled, clean with suitable laboratory
detergent and water. If the spilled liquid contains potentially infectious
agents, clean the affected area first with laboratory detergent and water, and
then with 1% (v/v) sodium hypochlorite.
Customer-Supplied Reagents and Equipment**
Disposable powder-free gloves
Benchtop microcentrifuge
Micropipettes
RNase-/DNase-free, sterile filter tips and pipettes
Vortex
96%–100% Molecular Biology Grade Ethanol
Sterile, RNase-/DNase-free tubes
PROTOCOL
This protocol describes the method for RNA extraction from EVs purified using
qEV columns.
Prior to RNA extraction:
Store samples at -80°C.
Prepare a working concentration of Wash Solution A by adding 90 mL of 96%–100%
ethanol to the supplied bottle (containing 38 mL of concentrated Wash Solution
A) to give a final volume of 128 mL.
Ensure that all solutions are at room temperature and that Lysis Buffer
A is warmed to 60°C for 20 minutes prior to use. If precipitates are present,
mix all solutions well until the solutions become clear.
Thaw samples on ice prior to RNA extraction.
For successful RNA extraction, the following practices are recommended:
Perform all centrifugation steps at room temperature using appropriate tubes.
n.b., Spin columns should not be used with vacuum apparatus.
If any solutions do not pass through the spin columns within the specified
centrifugation time, spin for an additional 1–2 minutes until the solution
completely passes through the column. Do NOT exceed the centrifugation speed.
After applying the Elution Solution A, allow to stand for 2 minutes
prior to centrifugation. This is an essential step in maximising yield. Do not
skip or shorten this step.
4.1 Procedure
If using the qEV Concentration Kit, separate the EV-bound Nanotrap® particles
from the supernatant by centrifuging the particle-sample suspension at 16,800
x g for 10 minutes at room temperature. Without disturbing the Nanotrap® EV
particle pellet, remove the supernatant (either transfer to a clean tube for
analysis of non-EV components of the sample, or discard). The remaining
Nanotrap® EV particle pellet is now ready for the RNA extraction procedure.
-
To your chosen volume of qEV isolate, add 1.5 volumes of Lysis Buffer A and 0.2 volumes of Lysis Additive B. This will be your Lysed EV Solution.
E.g., if you have 200 µL of qEV isolate, add 300 µL of Lysis Buffer A and 40 µL of Lysis Additive B, for a total Lysed EV Solution volume of 540 µL.
n.b., When beginning with an EV pellet such as the Nanotrap® EV particle pellet, add 250 µL of Lysis Buffer A and 20 µL of Lysis Additive B directly to the EV pellet to produce your Lysed EV Solution. -
Mix the Lysed EV Solution well by vortexing for 10 seconds, then incubate at room temperature for 20 minutes.
-
Following incubation, add 0.93 volumes (using the volume of the Lysed EV Solution as a reference volume) of 96%-100% ethanol to Lysed EV Solution and mix well by vortexing for 10 seconds. This is your Lysed EV Ethanol Solution.
E.g., to 540 µL of Lysed EV solution, add 502.2 µL of ethanol for a final volume of 1042.2 µL. -
Transfer up to 700 µL of the Lysed EV Ethanol Solution into the mini spin column. Centrifuge for 1 minute at 3,300 x g. Discard flowthrough.
-
Repeat step 4 until all Lysed EV Ethanol Solution has been run through the mini spin column.
-
Apply 600 µL of Wash Solution A to the column and centrifuge for 30 seconds at 3,300 x g, then discard flowthrough.
-
Repeat step 6.
-
Spin the empty column for 1 minute at 13,000 x g to remove any remaining liquid. Discard collection tube and transfer the spin column into a fresh 1.7 mL elution tube.
-
Apply a chosen volume of Elution Solution A between 50 and 100 µL to the column and centrifuge for 1 minute at 400 x g followed by a further 2 minutes at 5,800 x g.
A second elution can be performed if deemed necessary to further maximise
recovery. For this, take the eluate from step 9 (i.e., the same Elution
Solution A that has already passed through the column) and re-apply it to the
column. Incubate the column for 2 minutes at room temperature and centrifuge
to re-collect as in step 9. Do not use new Elution Solution A for this
optional step if you wish to increase concentration as this will result in a
dilution.
Following extraction, RNA should be stored at -80°C until it is required for
downstream applications.
FREQUENTLY ASKED QUESTIONS
What happens if I use the wrong centrifuge speed?****
– Centrifuging the mini spin columns at a speed higher than recommended may affect RNA yield. Centrifuging at a speed lower than recommended will not affect RNA yield but may increase the elution time.
What happens if I perform the centrifugation at the wrong temperature?****
– We recommend performing all centrifugation steps at room temperature; however, centrifugation at 4 °C will not adversely affect the kit performance.
How does Elution Solution A volume impact upon RNA?****
– Eluting your RNA in high volumes will increase the yield but the resulting sample will have a low concentration of RNA. Eluting in small volumes will increase the concentration but will reduce the overall yield.
What happens if I forget to do a dry spin before my final elution step?****
– Purified RNA will be contaminated with the Wash Solution A which may reduce the quality of the purified RNA and interfere with downstream applications.
Why do my samples show a low RNA yield?****
– The amount of RNA contained within EVs is very small and can vary depending on the sample that they were isolated from and purification methods used. The yield of RNA can be increased by increasing the amount of sample input. For optimal and standardised results, the qEV RNA Extraction Kit should be used in combination with qEV columns and the qEV Concentration Kit.
Why is the A260/280 ratio of the purified RNA not 2.0?****
– Measurement of A260/A280 ratios are inaccurate at low RNA concentrations, like the ones you are likely to see from EV-RNA isolation. This is reflected in a low A260/A280 value and is acceptable for downstream analysis. If the A260/A280 value is high, this indicates contamination. In this case, run the eluted RNA through the extraction procedure again from Step 3 onwards. This may reduce yield but should increase purity.
What happens if I use a different elution buffer?****
– If an elution buffer other than the one provided in the kit was used, it may interfere with downstream applications. Common components that are known to interfere are high concentrations of salt (including EDTA), detergents and other denaturing agents. Check the compatibility of your elution buffer with the intended use.
OTHER RESOURCES
Refer to the Safety Data Sheet for classification and labelling of hazards and
precautionary statements.
The Safety Data Sheet for the qEV RNA Extraction Kit is located at
support.izon.com/safety-datasheets
For more information or to contact a customer support representative please
visit support.izon.com
Rev H
www.izon.com
References
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