64×10ml Swing Buckets Rotor

The NGS Cell Free DNA Library Prep Kit (illumina and MGI Platforms) was developed for the construction of high quality cell-free DNA (cfDNA) libraries using 1 ng to 50 ng of cell-free DNA as input. The kit has a simple work flow and a fast procedure. Multiplexing of the cell free DNA library is possible based on the index type.

NGS Cell Free DNA Library Prep Kit Workflow

The main source of cell-free DNA (cfDNA) is derived from apoptotic hematopoietic cells in blood and found in the plasma. The length of the cfDNA is about 150-200 bp in length. Circulating tumor DNA (ctDNA) derived from malignant tumors is a part of cfDNA. Both cfDNA and ctDNA can be used as a noninvasive biomarker since it offers a better approach in comparison to invasive tissue biopsies.

NGS has been used for cfDNA and ctDNA sequencing in the field of liquid biopsy as it provides a whole genome level of molecular profiling. One of the hurdles for cfDNA sequencing is the difficulty of library preparation from the limited amount of cfDNA obtained from plasma. Our cell-free NGS kit makes it easy to get enough libraries from limited input in just 1.5 hours.

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

Non-index (Cat.# 30029): Libraries do not have index.

Index(Cat.# 30031): Each of our index primers contains a unique 6-base index sequence that can be used for sample identification. Total 48  library multiplexing is possible. Index information can be downloaded here.

Unique dual index (Cat.# 30033): Cell-free DNA library multiplexing up to 96 samples is possible with the unique dual indexes. We have developed a Four-Base Difference Index System. The system have at least 4 bases different from each other in the 8 bases index length. The primers effectively minimize sequencing errors such as mis-assignment, index hopping, index contamination etc. Index information can be downloaded here.

Indexes are available for the MGI platform kits (Cat.# 34031).

Kit advantages:

• Fast and simple protocol
  • Hands-on time is around 10 minutes
  • The total protocol time is only 1.5 hours
• Easy procedure based on:
  • Ready-to-use master mix for easy setup of reactions
  • Less reaction components to simplify bench work
• Less magnetic beads used for cleanup procedures: This can reduce more than 50% of the beads consumption
• Guaranteed high library conversion efficiency
• Low input cell-free DNA: From 1 ng to 50 ng

Comparison of library conversion efficiency with different samples under the same condition.

Comparison of library yield with different samples under the same condition.

The NGS Cell Free DNA Library Prep Kit (illumina and MGI Platforms) was developed for the construction of high quality cell-free DNA (cfDNA) libraries using 1 ng to 50 ng of cell-free DNA as input. The kit has a simple work flow and a fast procedure. Multiplexing of the cell free DNA library is possible based on the index type.

Opentrons Flex Magnetic Block (GEN 1) is a 96-well magnetic well plate holder containing static ring magnets, specifically designed for use with the Flex Robot. Use it with the Flex Gripper and skirted 96-well PCR or deep-well plates to facilitate quick and efficient separation using magnetic beads. This module is not compatible with the OT-2.

Opentrons Flex Magnetic Block (GEN 1) is a 96-well magnetic well plate holder containing static ring magnets, specifically designed for use with the Flex Robot. Use it with the Flex Gripper and skirted 96-well PCR or deep-well plates to facilitate quick and efficient separation using magnetic beads. This module is not compatible with the OT-2.

Overview

• Isolate all sizes of circulating and exosomal RNA, including microRNA
• Isolate all sizes of circulating DNA from plasma and serum samples
• Isolate viral and bacterial DNA and RNA
• Versatile plasma and serum input volumes (10 µL – 200 µL)
• No phenol extractions
• Bind and elute all RNA irrespective of size or GC content, without bias
• Concentrate circulating RNA, exosomal RNA and cell-free circulating DNA into a flexible elution volume ranging from 10 µL to 25 µL
• Isolate inhibitor-free nucleic acids
• Purify high-quality RNA and DNA in 30 minutes
• Purified RNA is suitable for a variety of downstream applications, including Small RNA Sequencing. Find out more information on Norgen’s NGS services
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

Norgen’s Plasma/Serum RNA/DNA Purification Mini Kit provides a fast, reliable, reproducible and simple procedure for the sequential isolation of circulating RNA, exosomal RNA and Cell-Free Circulating DNA (cfc) from the same Plasma/Serum sample. It can sequentially isolated RNA and DNA from small plasma/serum input ranging from 10 µL to 200 µL. The kit is designed to isolate all sizes of circulating RNA, including microRNA, all sizes of exosomal RNA as well as all sizes of cfc-DNA from fresh or frozen plasma or serum samples. Norgen’s Plasma/Serum RNA Purification Kit provides a clear advantage over other available kits in that it does not require Phenol/Chloroform or any Protease treatments for the isolation of plasma/serum RNA or DNA . RNA and DNA can be eluted into a flexible elution volume ranging from 10 µL to 25 µL. Purified RNA can be used in a number of sensitive downstream applications including reverse transcription qPCR, reverse transcription PCR, NGS, Northern blotting, RNase protection and primer extension, and expression array assays. Purified DNA can be used in a number of sensitive downstream applications including PCR, qPCR, methylation-sensitive PCR and Southern Blot analysis.

Background

Typical yields of free-circulating, exosomal RNA and cfc-DNA vary depending on the input sample, as the amount of RNA and/or DNA present in plasma and serum will depend upon the health status of the individual.  Normally, the RNA/DNA yield from plasma or serum is highly variable (range from 1 to 100 ng/mL). Variability is also observed between samples collected from the same donor at different times during the day. This kit is suitable for the isolation of RNA/DNA from serum or plasma prepared from blood collected on either EDTA or Citrate. Plasma samples prepared from blood collected on heparin should not be used as heparin can significantly interfere with many downstream applications such as RT-PCR.

Details

Supporting Data

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 Note: Do not exceed the recommended sample input volume of 200 µL.

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.