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View additional product information for MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit - FAQs (A36716)
23 product FAQs found
There are two analysis workflows available:
1. Oncomine TagSeq Lung v2 Liquid Biopsy - w2.0 - Single Sample: Detects and annotates low-frequency variants including SNPs/Indels (down to 0.1% limit of detection), Fusions, and CNVs from targeted nucleic acid libraries (DNA & RNA) from the Oncomine Lung Cell‑Free Total Nucleic Acid Research Assay. This is compatible with DNA & RNA purified from cell-free total nucleic acids.
2. Oncomine TagSeq Lung v2 Tumor - w2.0 - Single Sample: Detects and annotates low-frequency variants including SNPs/Indels (down to 0.5% limit of detection), fusions, and CNVs from targeted nucleic acid libraries (DNA & RNA) from the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay. Due to deamination events caused by the FFPE process, the minimum alternative allele frequency is set to 0.3%. This makes it compatible with DNA & RNA purified from FFPE tumor tissue as well as fresh frozen tumor tissue.
Yes, there is a specific BED file and Hotspot file for the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay. Please contact your local Field Application Specialist (FAS) or Clinical Application Consultant (CAC) to request the BED files.
Through the use of Tag Sequencing technology, low limits of detection (LOD) can be achieved for different variant types*:
- For SNVs/short indels, an LOD of 0.1% can be achieved with sensitivity of ˜90% and specificity of >99%
- For fusions & MET exon skipping, an LOD of 1% can be achieved with sensitivity of >90% and specificity of >99%
- For MET CNV target, detection as low as 1.2-fold amplification can be achieved with sensitivity of >90% and specificity of >99%
*Sensitivity and specificity for each variant type were determined using a collection of contrived positive samples and cfNA isolated from normal healthy donors.
No, this assay uses Tag Sequencing technology. Cell-free DNA (cfDNA) and cell-free RNA (cfRNA) are found at extremely low concentrations in the plasma fraction of whole blood. Because of this low prevalence, Taq Sequencing technology is utilized in this assay. The technology attaches unique molecular tags to the gene-specific primers. After amplification, the tagged molecules are grouped based on the tags. Groups containing the same mutant variant 80% of the time or greater will be called positive. Using the Tag technology, groups that contain random errors generated through the library construction/sequencing process are removed.
Use cell-free total nucleic acid (cfNA) extracted using a method optimized for cfNA isolation from plasma. We recommend the MagMAX Cell Free Total Nucleic Acid Isolation Kit (Cat. No. A36716). You can expect 5-50 ng of cfDNA and 5-100 pg of cfRNA from 10 mL blood research sample collected in a K2 EDTA blood collection tube (Cat. No. ??)
We recommend using the fluorescence-based Qubit dsDNA HS Assay Kit (Cat. No. Q32851) for quantification of cfNA and cfDNA samples. Spectrophotometric quantification methods are not recommended, because they are not reliable when the nucleic acid concentration is low. Use of these methods can lead to gross overestimation of the concentration of sample, under seeding of the target amplification reaction, and low library yields. There isn't a good absolute quantitation method for cfRNA.
We recommend using the Tag Sequencing BC Set 1-24 (Cat. No. A31830) and Tag Sequencing BC Set 25-48 (Cat. No. A31847) to generate barcoded libraries for multiplexed templating and sequencing.
We recommend sequencing the libraries on the Ion S5 or Ion S5 XL systems. The assay has been developed and verified for use on the the Ion 510 & Ion 520 & Ion 530 Kit - Chef, which requires Torrent Suite Software 5.4 or higher. Performance has been demonstrated using the Ion 540 Kit - Chef which requires Torrent Suite Software 5.2 or higher.
Here are the components of the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay:
- cfDNA Library PCR Master Mix
- Low TE Buffer
- Lung cfTNA Panel
- cfDNA Library Primer P1
- Tag Sequencing BC1
- SuperScript VILO Master Mix
It contains a single pool of multiplex PCR primers.
1 sample can be multiplexed on an Ion 318 chip, up to 6 samples can be multiplexed on an Ion 530 chip, and up to 24 samples can be multiplexed on an Ion 540 chip
The number of reads needed to detect single nucleotide variants (SNVs) for each library is ˜2.5 million. This determines hpw many samples can be multiplexed on a chip.
K2 EDTA blood collection tubes are preferred and can be purchasd from any major lab supplier. You can also use Cell-Free DNA BCT tubes from Streck (Cat. No. 218962).
For the best results, we recommend using plasma fraction from whole blood with minimal-to-low level of hemolysis. To prevent hemolysis during blood collection, please follow guidelines provided here (http://blog.fisherbioservices.com/avoiding-hemolysis-in-blood-sample-collection-and-processing blog). The Oncomine Lung Cell-Free Total RNA (cfNA) Research Assay is compatible with FFPE samples.
Note: Plasma samples with minimal-to-mild hemolysis are recommended to achieve minimal SNV false positives.
Yes, reverse transcription using the SuperScript VILO Master Mix is needed to convert cell-free RNA (cfRNA) into cDNA to enable structural variant detection (gene fusions and exon skipping).
The Oncomine Lung Cell-Free Total Nucleic Acid Research Assay contains sufficient reagents to prepare 8 libraries from cell-free total nucleic acid.
The input amount range is 1-50 ng and the recommended input amount is 20 ng. There is a high primer dimer peak when using <5 ng input. The higher input will shift the family size distribution (i.e., more reads will be needed to call out a variant). LOD (limit of detection) for SNV/small indels calls and fusion detection is more sensitive to the input amount whereas LOD for CNV calls is only slightly impacted by the input amount.
The Oncomine Lung Cell-Free Total Nucleic Acid Research Assay is part of a complete solution to detect lung tumor-derived cell-free DNA and RNA (cell-free total nucleic acid; cfNA) isolated from the plasma fraction of whole blood. This assay enables the analysis of:
- Hotspot genes (SNVs) and short indels: ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53 (˜168 hotspots covered). These genes have been identified as frequently mutated in non-small cell lung cancer (NSCLC).
- Gene fusions: ALK, RET, ROS1
- MET exon 14 skipping
- Copy number gene (CNV): MET
cfRNA concentrations in plasma are low, and the conformation/length is not compatible with the dye in the Qubit RNA HS assay. We recommend using an m1 TaqMan assay with an appropriate amplicon length to detect cfRNA, such as Hs_99999905_m1 GAPDH (122 bases), Hs99999903_m1 ACTB 171 bases or another m1 assay target that is appropriate for your sample. The m1 designation indicates that the probe spans an exon-exon boundary ensuring that signal is only generated from a template with correctly spliced exons. The assay will not detect signal from the cfDNA that is also present in the eluate. We recommend using SuperScript VILO Master Mix (Cat. No. 11755050) for the RT step and the reaction volume for this step can be scaled down to 10 µL total, using 2 µL of purified cfNA input. We recommend performing the qPCR reaction according to the protocol for TaqMan Universal Master Mix II, no UNG (Cat. No. 4440040), with sample input up to 10% of the total reaction volume.
The Qubit Fluorometer cannot discriminate between cell-free DNA and gRNA, so running an Agilent High Sensitivity DNA analysis chip is highly recommended.
Yes, the MagMAX Cell-Free Total Nucleic Acid Isolation Kit does isolate miRNA.
No, Proteinase K treatment is required to lyse the exosomes, which contain most of the cell-free RNA in plasma samples; it also degrades proteins.
NGS, qPCR, and ddPCR are all compatible with the MagMAX Cell-Free Total Nucleic Acid Isolation Kit. The kit is specifically designed for Ion Torrent liquid biopsy panels (Oncomine Lung Cell-Free Total Nucleic Acid Research Assay, Cat. No. A35864) that require cell-free DNA as well as cell-free RNA. The minimum yield requirement for these NGS panels is 2 ng/µL, which is why the protocol includes a rebinding step that allows for lower elution volume (15 µL) and yields a more concentrated nucleic acid.
For NGS applications, we recommend using K2EDTA tubes. For other applications, Streck Cell-Free DNA Blood Collection Tubes can also be used.