Overview

• 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

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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

Introduction

This Kit is designed forpurification of high quality circulating DNA (cfDNA) from 1-6ml cell-free bodyfluids (such as plasma, serum). The purified DNA is suitable for direct use indownstream applications such as PCR, real-time PCR, Biochip analysis and NGS.

Details

Specifications

Features	Specifications
Main Functions	Isolation circulating DNA from 1-6ml plasma, serum, body fluids
Applications	qPCR, NGS, etc.
Purification technology	Magnetic beads technology
Process method	Manual or automatic
Sample type	Serum, plasma
Sample amount	1-6ml
Elution volume	≥50μl
Time per run	≤60 minutes

Principle

This product is based on the purification method of high binding magnetic particles. The sample is lysed and digested under the action of lysate and protease. DNA is released into the lysate. After adding magnetic particles and binding solution, DNA will be adsorbed on the surface of magnetic particles, and impurities such as proteins will be removed without adsorption. The adsorbed particles were washed with washing solution to remove proteins and impurities, washed with ethanol to remove salts, and finally DNA was eluted by elution buffer.

Advantages

• Economy – less than 50% of the price of Qiagen and other imported products
• Automatic – without labour
• High purity – OD 260 / 280 : 1.7-1.9, OD 260 / 230 :1.5-2.0

Kit Contents

Contents	IVD5435
Purification Times	50
MagPure Particles F	14 ml
Carrier RNA	310 μg
Proteinase K	240 mg
Protease Dissolve Buffer	15 ml
Buffer SDS	15 ml
Buffer MLK	500 ml
Buffer MAW1	250 ml
Buffer MW2*	50 ml
Elution Buffer	60 ml

Storage and Stability

Proteinase K, Carrier RNA and MagPure Particles F should be stored at 2–8°C upon arrival. However, short-term storage (up to 12 weeks) at room temperature (15–25°C) does not affect their performance.Theremaining kit components can be stored dry at room temperature (15–25°C) and are stable for at least 18 months under these conditions.The entire kit can be stored at 2–8°C, but in this case buffers should beredissolved before use. Make sure that all buffers are at room temperature when used.

Experiment Data

This Kit is designed forpurification of high quality circulating DNA (cfDNA) from 1-6ml cell-free bodyfluids (such as plasma, serum). The purified DNA is suitable for direct use indownstream applications such as PCR, real-time PCR, Biochip analysis and NGS.

Product Description

High Sensitivity RNA Amplification With Improved Detection Isothermal Kit

Product Detail

Applicable equipment:

It is recommended to use the Isothermal fluorescence detector developed by Amp-future, which is also suitable for fluorescence quantitative PCR apparatus with market known brands.

Kit Storage and term of Validity

Storage term: stored at ≤-20℃,keep away from light, avoid heavy weight and repeated freezing and thawing.

Term of Validity: 14 months

Isothermal nucleic acid Principle Summary

This kit is based on a rapid nucleic acid amplification technology at room temperature and constant temperature: at room temperature and constant temperature (generally 39℃~42℃), with the help of auxiliary proteins and single-strand binding proteins,the recombinase and primers form a complex;Source search and combine the target homology domain, at this time,a D-loop region is formed at the homology position and strand exchange begins;along with the dissociation of the recombinase from the complex,the polymerase also binds to the 3′ end of the primer and begins chain extension. At the same time, relying on the function of exonuclease, adding specific molecular probes designed according to the template, and using fluorescence monitoring equipment can realize real-time monitoring of the amplification process of the target fragment.

Isothermal nucleic acid Product Features

1/ High sensitivity and specificity, short reaction time.

2/ The reagent form is freeze-dried, stable and easy to operate.

Technical Parameters:

Parameters	Details
Product Name	DNA Isothermal Amplification Kit EXO
Manufacturer	Amp-future
Storage Temperature	-20°C
Kit Components	Enzymes, Buffers ,Reagents
Packaging	48 Tests/box
Detection Limit	500-1000copies/µL
Shipping	ICE
Test Time	5-20mins

Isothermal nucleic acid Applications

Suitable for DNA isothermal rapid amplification kit(fluorescent type)

Primer: Require pair of nucleotide primers with the length of 25-35 bp.

Fluorescent Probe:Require the suitable length is 46-52nt.

DNA fluorescent kit reaction temperature is 39 to 42℃ and time is 5-20 minutes.

Notes

1/ Please avoid nucleic acid contamination and set blank control during reaction due to the high sensitivity of the kit.

2/ Please take out the required quantity of MIRA reaction units for the experiment, and put the rest under storage conditions when performing the experiment.

It is recommended to use the Isothermal fluorescence detector developed by Amp-future, which is also suitable for fluorescence quantitative PCR apparatus with market known brands.