Norgen’s EXTRAClean Cell Culture Media Exosome Purification and RNA Isolation Kits constitute all-in-one systems for the purification of exosomes and the subsequent isolation of RNA from different cell culture media sample volumes. 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 it does not require any special instrumentation, protein precipitation reagents, extension tubes, phenol/chloroform or protease treatments. Moreover, the kits allows the user to elute into flexible elution volumes ranging from 50 μL to 100 μL. The purified RNA is free from any protein-bound circulating RNA and is of the highest integrity. The purified RNA can be used in a number of downstream applications including real time PCR, Sequencing based applications, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.
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| Sample Type | Cell-Free Cell Culture Media |
| Sample Volume Range | 20 ml to 35 mL |
| Size of RNA Purified | All sizes, including miRNA and small RNA (< 200 nt) |
| Elution Volume | 50-100 μL |
| Time to Complete 10 Purifications | 40-45 minutes |
| Average Yields | Variable depending on specimen |
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. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.
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.
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.
The HiPure Plant RNA Midi Kit provides fast purification of high-quality RNA from plants, cell, tissues, and yeast using silica-membrane spin columns with a binding capacity of 1mg RNA. There is no need for phenol / chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation or precipitation with isopropanol or LiCl. RNA purified using the RaPure Total RNA Purification System can be used for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.
Specifications
| Features | Specifications |
| Main Functions | Isolation total RNA from <1g simple plant sample without chloroform |
| Applications | RT-PCR, qPCR, Northern hybridization, second generation sequencing, nucleic acid protection, in vitro translation |
| Purification method | Midi spin column |
| Purification technology | Silica technology, DNA filtration technology |
| Process method | Manual (centrifugation or vacuum) |
| Sample type | Plant leaves, roots, stems, fruits, fungi, etc. |
| Sample amount | 0.5-1 g |
| Elution volume | ≥400μl |
| Time per run | ≤40 minutes |
| Liquid carrying volume per column | 4ml |
| Binding yield of column | 1mg |
The Kit isolates total RNA from up to 1g plant tissue. A short workflow enables RNA isolation with genomic DNA removal in less than 40 min. Samples are first lysed and homogenized. The lysate is passed through a DNA Mini column, ethanol is added to the flow-through, and the sample is applied to a RNA column. RNA binds to the membrane and contaminants are washed away. High-quality RNA is eluted in as little as 400µl water using the Kit.
Advantages
Kit Contents
| Contents | R415202 | D415203 |
| Purification Times | 20 Preps | 100 Preps |
| HiPure DNA Midi Column II | 20 | 100 |
| HiPure RNA Midi Columns II | 20 | 100 |
| 15ml Collection Tubes | 40 | 200 |
| Buffer RLC | 120 ml | 550 ml |
| Buffer PRC1 | 120 ml | 550 ml |
| Buffer RW1 | 70 ml | 400 ml |
| Buffer RW2* | 20 ml | 100 ml |
| RNase Free Water | 30 ml | 120 ml |
Storage and Stability
The Kit can be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions. Make sure that all buffers are at room temperature when used. During shipment, crystals or precipitation may form in the Buffer RLC/PRC1. Dissolve by warming buffer to 37°C.
1. When dealing with woody or uncommon samples, R4150 is recommended first. R4150 contains two polysaccharide/polyphenol lysis buffer, which is the most universal product.
2. R4151 is recommended for handling common economic crop samples for the first time. Strong lysis solution can be used to process easy-extraction samples. The amount of corn or rice leaves samples can reach up to 300mg.
3. R4165 adopts CTAB/chloroform method, which can also handle a large number of difficult-to-extraction plants, but requires contact with chloroform substitutes, which is less safe than other kits. This kit uses DNase Ⅰ to remove DNA, which is also a good choice for extracting polysaccharide/polyphenol-rich plant samples.
4. R4014 is recommended for fruit/starch plant samples, which uses improved trizol pre-treatment, single column operation and is more economical.
Select the right purification kit to get impactful results：
Ⅰ. Purchase guide of Magen kits based on sample
Ⅱ. Cross purchase guide for Magen kits vs Other brands
For any customized product or OEM service, please contact us.
The HiPure Plant RNA Midi Kit provides fast purification of high-quality RNA from plants, cell, tissues, and yeast using silica-membrane spin columns with a binding capacity of 1mg RNA. There is no need for phenol / chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation or precipitation with isopropanol or LiCl. RNA purified using the RaPure Total RNA Purification System can be used for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.