Overview

• Detection kits for XMRV
• Available in TaqMan format for analysis

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus. The virus was first described in 2006 and has since been isolated from human biological samples. XMRV belongs to the family Retroviridae and the genus gammaretrovirus. It has a single-stranded RNA genome that replicates through a DNA intermediate. The virus gets its name due to its close relationship with the murine leukemia viruses (MuLVs). The viral genome is approximately 8100 nucleotides in length and is 95% identical with several endogenous retroviruses of mice. While gammaretroviruses have well-characterized oncogenic effects in animals, they have not been shown to cause human cancers. However, XMRV was recently discovered in human prostate cancers and is the first gammaretrovirus known to infect humans. In addition to prostate cancer, a possible association with chronic fatigue syndrome has been reported, however it has yet to be established whether XMRV is a cause of this disease.

The causal role of XMRV in cancer has yet to be established and the virus does not appear to be capable of transforming cells directly. In prostate cancer, XMRV protein has been found in tumour-associated but nonmalignant stromal cells, but not in the actual prostate cancer cells. This raises the possibility that the virus may support tumorigenesis. In other studies, XMRV proteins and nucleic acids were found in malignant cells.

Details

Supporting Data

Figure 1 / 3

Previous

Next

Click for expanded view

Storage Conditions and Product Stability
All kit components can be stored for 1 year after the date of production without showing any reduction in performance.

All kit components should be stored at -20°C upon arrival. Repeated thawing and freezing (> 2 x) of the Master Mix and Positive Control should be avoided, as this may affect the performance of the assay. If the reagents are to be used only intermittently, they should be frozen in aliquots.

Component	Cat. TM34750 (100 preps)	Cat. TM34710 (100 preps)
MDx TaqMan 2X PCR Master Mix	2 x 700 μL	–
XMRV Primer & Probe Mix	280 μL	280 μL
XMRV Positive Control	150 μL	150 μL
Nuclease-Free Water (Negative Control)	1.25 mL	1.25 mL
Product Insert	1	1

Overview

• Isolate and extract high quality & quantity miRNA in 30 minutes
• Two column kit- eliminate large RNA on the first column and capture microRNA on a second column
• Elute microRNA in 25 µL ready for miRNA profiling
• Process a wide range of samples- cell, tissue, bacteria, bodily fluids, etc.
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

This kit provides a rapid method for the isolation and purification of small RNA molecules (< 200 nt) from cultured animal cells, small tissue samples, bacterial cells, plants, and blood.

Two columns are provided with this kit. The first column captures large RNA, while the small RNA are captured on a second column and are eluted concentrated in 25 µL of nuclease-free water.

The small RNA can be used in various downstream applications relating to miRNA profiling, gene regulation, and functional analysis. The eluted RNA is ready for RT-qPCR, microarrays, and NGS applications.

Background

These small RNAs include regulatory RNA molecules such as microRNA (miRNA) and short interfering RNA (siRNA), as well as tRNA and 5S rRNA. Small RNA molecules are often studied due to their ability to regulate gene expression. miRNAs and siRNAs are typically 20-25 nucleotides long and regulate gene expression by binding to mRNA molecules and affecting their stability or translation.

Details

Supporting Data

Figure 1 / 4

Previous

Next

Click for expanded view

Kit Specifications
Maximum Column Binding Capacity	Up to 50 μg RNA
Maximum Column Loading Volume	650 μL
Minimum Elution Volume	20 μL
Size of RNA Purified	All sizes, including small RNA < 200 nt
Time to Complete 10 Purifications	25 minutes
Maximum Amount of Starting Material: Animal Cells Animal Tissues Bacteria Plant Tissues Blood	3 x 106 cells 5-25 mg 1 x 109 cells 50 mg 100 μL

 Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.

Component	Cat. 21300 (25 preps)
Buffer RL	40 mL
Wash Solution A	38 mL
Elution Solution A	6 mL
Large RNA Removal Column	25
microRNA Enrichment Column	25
Collection Tube	50
Elution Tubes (1.7 mL)	50
Product Insert	1

cfDNA Purification Kit (Magnetic Beads)

The cfDNA Purification Kit (Magnetic Beads) was developed for cell free DNA (cfDNA) enrichment by separating genomic DNA contamination from isolated cfDNA samples.

Many diagnostic technologies for detection of disease signals in cfDNA begin with isolation and purification of DNA from liquid biopsy that include urine, plasma, cerebrospinal fluid. The most widely explored biotechnology is assays used to detect cancer-derived plasma cfDNA. Silica-based magnetic bead cfDNA isolation kits can reliably extract total DNA from plasma, but typically yield a large variation in cfDNA that includes the presence of genomic DNA that often depends on tumor stage, tumor size, or healthy status individuals. Most of the commercial cfDNA isolation kits can’t specifically recover the cfDNA while leaving the high molecular weight genomic DNA behind. The presence of genomic DNA can lead to decreased sensitivity or inconsistent results in downstream applications such as next-generation sequencing (NGS), PCR, QPCR, and digital PCR etc.

Therefore, an additional purification step to enrich cfDNA before downstream methods helps to improve signal from fragments that originate from cancer cells. A proportion of cancer-derived cfDNA fragment signals are below 100 bp and are often not detectable except by qPCR or single-stranded DNA based library preparation for NGS (1, 2, 3). Furthermore only 1% of cancer-derived fragments are found above 400 bp (1, 2). Capture of size-selected fragments between 90-150 bp improved detection of cancer by 2-4 fold (4). Furthermore, TF-bound and protected cfDNA fragments are also being investigated for active cancer-specific signals down to 35-80 bp (5, 6).

This kit uses Dual Solid Phase Reversible Immobilization (SPRI) technology for cfDNA purification. Most Dual SPRI procedures do NOT recover fragments below 100 bp. The kit can be used for the enrichment of cfDNA isolated from liquid biopsies, plasma, serum, and urine. The kit separates cfDNA (50-500 bp) and genomic DNA, and recovers of 90% of the cfDNA without the high molecular weight genomic DNA with high efficiency. Fragments at 500 bp and above may also be retained. Both the 50-500 bp and >500 bp DNA fractions can be used for downstream applications such as single-stranded or double stranded NGS library prep, qPCR, ddPCR, and other methods.

Features

• Separation of cfDNA and genomic DNA; Recovery of both types of DNA
• Recovery of cfDNA (50-500 bp)
  • As short as 50 bp can be recovered
• Recovery of high molecular weight genomic DNA
• Removal of unwanted components and other impurities
• Automation friendly

---

Examples of cfDNA purification. Both cfDNA and genomic DNA can be recovered separately.

---

The range of recovered small DNA fragments is from 50 to 500 bp. The input DNA are mixtures of sheared small DNA fragments and intact genomic DNA. The ratios of sheared DNA fragments versus genomic DNA are indicated.

---

Recovery rates of cfDNA and genomic DNA.

Many diagnostic technologies for detection of disease signals in cfDNA begin with isolation and purification of DNA from liquid biopsy that include urine, plasma, cerebrospinal fluid. The most widely explored biotechnology is assays used to detect cancer-derived plasma cfDNA. Silica-based magnetic bead cfDNA isolation kits can reliably extract total DNA from plasma, but typically yield a large variation in cfDNA that includes the presence of genomic DNA that often depends on tumor stage, tumor size, or healthy status individuals. Most of the commercial cfDNA isolation kits can’t specifically recover the cfDNA while leaving the high molecular weight genomic DNA behind. The presence of genomic DNA can lead to decreased sensitivity or inconsistent results in downstream applications such as next-generation sequencing (NGS), PCR, QPCR, and digital PCR etc.