Overview

• Ensure optimal results for sensitive applications like NGS
• Up to a tenfold increase in microRNA mapping during sequencing runs to reduce costs
• The purified exosomal RNA is free from any circulating RNA-binding proteins.
• No phenol extractions, Proteinase K treatment, nor carrier RNA.
• No time-consuming ultracentrifugation, filtration nor special syringes are required.
• Concentrate isolated RNA into a flexible elution volume ranging from 50 μL to 100 μL.
• Purification is based on EXTRAClean spin column chromatography that uses Norgen’s proprietary separation matrix.

Norgen’s EXTRAClean Plasma/Serum Exosome and Free-Circulating RNA Isolation Kit constitutes an all-in-one system for the purification of exosomes and the sequential isolation of RNA and free-circulating RNA from different plasma/serum sample volumes ranging from 50 μL and up to 10 mL. The purification is based on spin column chromatography that employs Norgen’s proprietary resin. The EXTRAClean columns undergo stringent processing and rigorous quality control measures to minimize contamination traces, ensuring optimal results for sensitive applications such as NGS. The kit is designed to isolate all sizes of RNA, including microRNA. The kit provides a clear advantage over other available kits in that they do not require any special instrumentation, protein precipitation reagents, extension tubes, phenol/chloroform or any protease treatments. Moreover, the kit allows the user to elute into a flexible elution volume ranging from 50 μL to 100 μL. The RNA isolated from the purified exosomes is free from any protein-bound circulating RNA and is of the highest integrity. Moreover, the free-circulating, protein-bound, RNA is free from any exosomal RNA. The purified RNA can be used in a number of downstream applications including real time PCR, NGS application, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.

Details

Supporting Data

Figure 1 / 9

Previous

Next

Click for expanded view

Kit Specifications (Spin Column)
Minimum Plasma Input	50 uL
Maximum Plasma Input	1 mL
Size of RNA Purified	All sizes, including miRNA and small RNA (< 200 nt)
Elution Volume	50-100 μL
Time to Complete 10 Purifications	35-40 minutes
Average Yields	Variable depending on specimen

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.

The NGS FFPE DNA Library Prep Kit (illumina and MGI Platforms) was developed for the construction of high quality FFPE DNA libraries using 10 ng to 400 ng of input DNA isolated from formalin-fixed, paraffin-embedded (FFPE) samples. The DNA damage caused by fixation makes it difficult to construct high quality libraries. Our kit has been optimized to repair damaged DNA in the reactions. Multiplexing of the NGS FFPE DNA Library is possible.

NGS FFPE DNA Library Prep Kit Workflow

FFPE samples are a great resource for biomedical research. However, the methods for fixation and condition of storage significantly damage the DNA in the samples.  Thus, the extraction of high quality DNA from FFPE samples is often a challenge.  Low yield and low quality of FFPE DNA usually are common because of the limited tissue material and the DNA degradation.

As a result, it is usually difficult to construct high quality NGS libraries from low amount and low quality of FFPE DNA. In order to address this issue, we have developed the NGS FFPE DNA Library Prep Kit to make high quality libraries from the low input of FFPE DNA samples.

Three index types are available for the NGS FFPE DNA Library Prep Kit of the illumina platform:

Non-index (Cat.# 30035): Libraries do not have index.

Index (Cat.# 30037): Each of our index primers contains a unique barcode DNA (6 bases long) that can be used to identify individual library. Multiplexing of libraries is up to 48 samples. Index information can be downloaded here.

Unique dual index (Cat.# 30039): FFPE DNA library multiplexing is possible with 96 samples based on the unique dual indexing system. Our unique Four–Base Difference Index System allows us to make indexes that have at least 4 bases different from each other in the 8-base index sequence. Our unique dual indexing primers can effectively remove NGS errors including index hopping, de-multiplexing errors, index cross-contamination, mis-assignments etc. The unique dual index primer set includes 96 pre-mixed unique pairs of i5 and i7 index primers. Index information can be downloaded here.

Indexes are available for the MGI platform kits (Cat.# 34037).

Kit advantages:

• Fast and simple protocol
  • Making libraries in just 1.5 hours
  • 10 minutes of hands-on time
• Easy procedure
  • Ready-to-use master mix to reduce the tedious mixing
  • Less reaction components to simplify the reaction setup
• Less magnetic beads needed for the purification steps: saving more than 50% of the expensive beads
• Low input FFPE DNA: From 10 ng to 400 ng

Comparison of library conversion efficiency under the same condition. Input DNA amount is 25 ng.

Comparison of library yield under the same condition. Input DNA amount is 25 ng.

The NGS FFPE DNA Library Prep Kit (illumina and MGI Platforms) was developed for the construction of high quality FFPE DNA libraries using 10 ng to 400 ng of input DNA isolated from formalin-fixed, paraffin-embedded (FFPE) samples. The DNA damage caused by fixation makes it difficult to construct high quality libraries. Our kit has been optimized to repair damaged DNA in the reactions. Multiplexing of the NGS FFPE DNA Library is possible.

Introduction

With the development of molecular biology, stool, a new non-invasive sample, has been widely used in the research of animal molecular genetics, population ecology, behavioral ecology and some intestinal disease diagnosis. Stool samples includes gut microbial DNA, food residue sample DNA, and alimentary tract exfoliated cell DNA.

The primary problem encountered when using stool sample for molecular biology research is the low content of exfoliated cells in the digestive tract and a certain degree of degradation of genetic material in stool. Another issue in molecular scatology research based on PCR is the presence of a large number of inhibitors in stool that can affect Taq enzyme activity, leading to downstream detection inactivation. These inhibitors include polysaccharides, plant polysaccharides, bile acids, bile salts, bile pigments, digestive juices, mucus, etc. Therefore, selecting appropriate extraction methods to obtain high-quality DNA is the key to successful downstream detection of stool DNA.

At present, the pretreatment methods used in the laboratory, such as phenol/chloroform extraction, cetyltrimethyl bromide (CTAB) lysis, and guanidine isothiocyanate lysis, lack universality in different species, and the success rate of extracting DNA for PCR amplification is also very low. The HiPure Stool DNA Kit provided by Magen Company has opened up a new approach for DNA extraction from stool samples with good universality, high cost-effectiveness, high yield and purification. The reagent kit adopts a unique solution system and inhibitory factor adsorbent, which can efficiently remove various impurities in stool samples. The purified DNA can be directly used for PCR, quantitative PCR and other applications.

This product allows rapid and reliable isolation of high-quality genomic DNA from various stool samples. Up to 100 mg soil samples can be processed in 60 minute. The system combines the reversible nucleic acid binding properties of HiPure matrix with the speed and versatility of spin column technology to eliminate PCR inhibiting compounds such as humic acid from soil samples. Purified DNA is suitable for PCR, restriction digestion, and next-generation sequencing. There are no organic extractions thus reducing plastic waste and hands-on time to allow multiple samples to be processed in parallel.

Details

Specifications

Features	Specifications
Main Functions	Isolation total DNA from 50-100mg stool samples
Applications	PCR, Southern Blot, enzyme digestion and NGS, etc.
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Stool
Sample amount	50-100mg
Yield	3-15μg
Elution volume	≥30μl
Time per run	≤60 minutes
Liquid carrying volume per column	750μl
Binding yield of column	100μg

Principle

Stool sample is homogenized and then treated in a specially formulated buffer containing detergent to lyse bacteria, yeast, and fungal samples. Humic acid, proteins, polysaccharides, and other contaminants are removed using our proprietary Absorber Solution. Binding conditions are then adjusted and the sample is applied to a DNA Mini Column. Two rapid wash steps remove trace contaminants and pure DNA is eluted in low ionic strength buffer. Purified DNA can be directly used in downstream applications without the need for further purification.

Advantages

• High purity – unique adsorbent can completely remove inhibitory factors
• High concentration – maximum extraction of total DNA from stool samples
• High recovery – DNA can be recovered at the level of PG
• Good repeatability – silica technology can obtain ideal results every time

Kit Contents

Contents	IVD3141
Purification Times	50 Preps
HiPure DNA Mini Columns II	50
2ml Collection Tubes	50
2ml Bead Tubes	50
Proteinase K	24 mg
Protease Dissolve Buffer	1.8 ml
Buffer SPL	40 ml
Buffer PCI	40 ml
Buffer AL	20 ml
Buffer GW1	22 ml
Buffer GW2	20 ml
Buffer AE	15 ml

Storage and Stability

Proteinase K and Buffer PCI 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. The remaining kit components can be stored 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 be redissolved before use. Make sure that all buffers are at room temperature when used.

With the development of molecular biology, stool, a new non-invasive sample, has been widely used in the research of animal molecular genetics, population ecology, behavioral ecology and some intestinal disease diagnosis. Stool samples includes gut microbial DNA, food residue sample DNA, and alimentary tract exfoliated cell DNA.