N-Boc-N-bis(PEG2-propargyl)

Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.

Whole genome bisulfite sequencing (WGBS) is the most effective method of DNA methylation analysis. The only limitation is the sequencing cost is very high because the whole genome is sequenced including all the non-methylated regions.

Reduced Representation Bisulfite Sequencing (RRBS) is the reduced representation of a smaller fraction of the methylated CpG sites. RRBS combines restriction enzyme digestion and bisulfite sequencing, and enriches the sequencing for methylated CpG sites. It is an efficient technology for estimate the whole genome methylation patterns at the single base level. Although this allows a higher coverage depth and reduces the sequencing cost, the limitation is only 10% of the methylated CpG sites are covered.

Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) is another whole genome enrichment technique used for selection of methylated DNA. Using antibodies against 5-methylcytosine, methylated DNA is enriched from whole genomic DNA via immunoprecipitation. 5-methylcytosine antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing. There are several drawbacks of MeDIP-Seq: 1. Low resolution (150~200 bp) as opposed to the single base resolution; 2. Non-specific interaction due to antibody specificity and selectivity. 3. Bias towards hypermethylated regions.

The Methylation Specific Bisulfite Seq (MSBS) Library Prep Kit (illumina platform) was developed for construction of NGS libraries for methylated CpG sites using bisulfite treated DNA (20 ng – 500 ng) as input. The kit enriches methylated CpG regions, thus significantly reduce the sequencing cost. The kit estimates the whole genome methylation patterns at the single base level since it is based on a bisulfite-seq technology.

It is known that bisulfite treatment of completed NGS libraries causes tremendous damage to the libraries. By using bisulfite treated DNA as input, the kit overcomes the significant library loss due to the bisulfite conversion. The kit contains a mixture of PCR polymerases that have high-fidelity amplification and uracil tolerance which is ideal for bisulfite treated DNA.

Methylation Specific Bisulfite Seq Library Prep Kit Workflow

Three index types are available for the kit:

Non-index (Cat.# 30101): Libraries do not have index.

Index (Cat.# 30102): Each primer contains a unique barcode sequence of 6 bases to identify the individual library. Library multiplexing capacity is up to 48 samples. Index information can be downloaded here.

Unique dual index (Cat.# 30103): The multiplexing of bisulfite sequencing library is up to 96 samples with unique dual indexes. We used a Four-Base Difference Index System to generate indexes that have at least 4 bases different from each other in the 8-base index. The index primers remove NGS errors including index cross-contamination, index hopping, reads mis-assignment etc. Index information can be downloaded here.

Methylation Specific Bisulfite Seq advantages

• • • Enrichment of methylated CpG sites
    • Single-base resolution
    • Low cost for sequencing
    • Fast
      • Total time: 1.5 hours
      • Hands-on time: 10 minutes
    • Simple workflow
    • Bisulfite treated DNA as input: From 20 ng to 500 ng

MSBS Library Prep Kit enriches CpG sites

High methylation regions and low methylation regions in human genome.

High methylation region in human genome.

Low methylation region in human genome.

Sequencing setting: Single-end 35 cycles (Read 1, 35 bases) recommended
To maximize the methylated CpG enrichment, we recommend to sequence the MSBS libraries with single end 35 cycles (read1, 35 bases). This is because the enriched methylated CpG sites are mainly located around the beginning of read 1 sequences. Shorter single end reads tend to have better methylated CpG enrichment.

Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.

Safety Features

1. User-Friendly Design: The chamber lid and tabletop are covered with heat-insulating resin materials, providing both protection against burns and an aesthetically pleasing appearance. The upward-opening flip-top door saves space, while the rounded corners prevent injury. The specially designed steam discharge and collection system minimizes steam burn risks and enhances operational convenience.

2. Interlock Mechanism and Safety Protection: The safety interlock system is designed to be more sensitive and secure. The chamber lid cannot be opened if there is pressure or steam inside the chamber. The lid can only be opened when the pressure gauge reads “0” MPa. The sterilizer will not start heating if the chamber lid is not properly locked. These features enhance the overall safety of the machine.

3. Over-temperature and Heating Protection: The system continuously monitors the temperature inside the sterilization chamber. If the temperature becomes too high or heating is abnormal, power will be automatically cut off to prevent overheating.

4. Automatic Fault Detection System: The machine performs an automatic self-check when powered on. If a fault occurs during operation, an audible alarm will sound, and a fault code will be displayed.

5. Lid Closure Check System: The system automatically detects whether the chamber lid is securely locked. If the lid is not properly locked, the sterilizer will not start.

6. Leakage, Over-current, and Short-Circuit Protection: If there is leakage in the heating elements or other components, or if over-current or short-circuit conditions occur, the system will automatically cut off power to prevent user injury.

7. Water Shortage and Independent Dry-Burning Protection: Equipped with a low water level sensor and independent dry-burning protection device, the system can monitor the water level in the chamber in real-time to prevent dry burning and avoid false readings. If the low water level sensor fails, the overheat and dry-burning protection system will automatically activate, cut off power to the heating ring, and sound an alarm to ensure safety.

8. Dual Pressure Protection: The system includes both a mechanical safety valve and an electronic over-pressure protection system. If pressure becomes abnormal, the system will relieve pressure and cut off power while sounding an alarm.

9. Back end Safety Testing: The system can be periodically tested for safety and protective measures.

10. Over-pressure Testing: Regular testing ensures that the instrument’s over-pressure protection system can automatically cut off power or that the safety valve can open to relieve pressure.

11. Performance Testing: The instrument’s safety performance has been tested by national authorized inspection agencies, and a test report is available.

Technical Parameters

Note: The upper limits for cooling lock door temperature and exhaust temperature are related to the boiling point.

Other Optional Accessories

1. Dot Matrix Printer: Capable of printing temperature, time, F0 value, and pressure data. USB data export and curve plotting.

2. Stainless Steel Basket with Base.

3. Pressure Sensor

4. Stainless Steel Bucket

5. Item Thermometer

6. 3Q Validation Adapter

Features and functions:

1. Large-diameter sterilization chamber：The 40 cm large-diameter sterilization chamber provides an exceptionally spacious environment to meet a wide range of sterilization needs.

2. Sterilization temperature：105-138℃.

3. Exhaust control ：After sterilization is complete, the exhaust valve can automatically open to release the exhaust at a pre-set temperature.

4. Verification interface: Provides temperature and pressure inspection ports for easy calibration.

5. Saturated steam monitoring：The system automatically monitors the expulsion of cold air to ensure a pure steam sterilization environment, guaranteeing optimal sterilization effectiveness.

6. Pressure performance：The chamber is designed to withstand a pressure of up to 0.35 MPa, and each unit is tested with a water pressure of up to 0.44 MPa. Advanced pressure protection ensures the safety load of the equipment.

7. Intelligent control system:The microcomputer chip-based intelligent control system monitors the temperature and pressure inside the sterilization chamber in real-time. The chamber lid can only be opened under safe temperature and pressure conditions. The system also provides fully automatic control of the sterilization process and allows for convenient maintenance, inspection, calibration, recording, and future upgrades.

8. Six preset programs:Includes six fixed programs for liquid sterilization and holding, solid sterilization and drying, agar melting and holding, and waste sterilization.

9. Memory storage system:Users can set their own parameters, create multiple programs, and access them at any time. The system allows for operation permissions to be set at three levels—user, administrator, and engineer—to facilitate effective use and management of the sterilizer.

10. Door-opening temperature: Each program can be set with a specific cooling lock door-opening temperature based on the thermal inertia of the sterilized items. This prevents users from opening the door when the items (especially liquid sterilized items) are still in an unsafe condition, thus avoiding burns.

11. Drying function: Thorough drying that meets CE standards, with a cooling lock mechanism.

12. One-touch inquiry function: Allows checking of current time, total number of sterilization cycles, fault occurrence time, and codes.

13. Scheduled start function: Allows you to set a start time so that the sterilizer automatically starts at the scheduled time.

Model:
HB65/ HB90/ HB110

Overview

• Isolate all sizes of circulating and exosomal RNA, including microRNA
• Versatile plasma/serum input ranges
• No phenol extractions
• No carrier RNA
• Bind and elute all RNA irrespective of size or GC content, without bias
• Concentrate circulating RNA and exosomal RNA into a flexible elution volume
• High quality, purified RNA is suitable for a variety of downstream applications, including Small RNA Sequencing. Find out more information on Norgen’s NGS services
• Compatible with Streck Cell-Free RNA BCT® Tubes
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

These kits provide a fast, reliable and convenient method to purify and concentrate high quality, high purity and inhibitor-free cell-free circulating and exosomal RNA using a convenient spin column method. These kits can purify RNA from fresh or frozen serum or plasma samples 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. The purified plasma/serum RNA is fully compatible with all downstream applications including PCR, qPCR, methylation-sensitive reverse transcription qPCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, expression array assays, and NGS.

Background

Plasma/Serum cell-free circulating RNA or exosomal RNA has the potential to provide biomarkers for certain cancers and disease states. 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 ascitic fluids, among other biological fluids. Evidence has been accumulating recently that these vesicles act as cellular messengers, conveying information to distant cells and tissues within the body. 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. 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 plasma or serum.

Plasma/Serum RNA Purification Mini Kit

This kit can purify RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate, from volumes ranging from 50 µL to 200 µL. The purified plasma/serum RNA is eluted in a flexible final volume of 10 µL to 25 µL.

Plasma/Serum RNA Purification Midi Kit

This utilizes a two column method, and can purify RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate, from volumes ranging from 250 µL to 1.5 mL. The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 50 µL to 100 µL.

Plasma/Serum RNA Purification Maxi Kit

This kit can purify RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate, from volumes ranging from 2 mL to 5 mL. The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 50 µL to 100 µL.

Isolate RNA after Purifying EVs and Exosomes

For Ultracentrifugation, Exoquick, and Filtration

Details

Supporting Data

Figure 1 / 13

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*This kit is suitable for the isolation of RNA from fresh or frozen 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.

**Please check page 7 for Average Plasma/Serum Yields and Common RNA Quantification Methods.

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.