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View additional product information for Qubit™ dsDNA Quantification Assay Kits - FAQs (Q32853, Q32854, Q32851, Q32850)
43 product FAQs found
Here are several suggestions:
1.View the raw fluorescence value (RFU) for the standards under “Check Standards” or “Check Calibration”. Confirm that the values for the samples fall between the values of the standards (or a little above the highest standard). If they do not, the sample is out of the accurate range of the assay. Refer to the confidence ranges for each assay in the product manuals. The readout in the assay will be to 2 significant figures instead of 3 if the assay sample is out of the high confidence range.
To bring the sample into the accurate range, dilute the sample or use more or less of it (for example, 10 µL instead of 2 µL if the sample reads low).
2.Check for temperature issues: The assay is temperature sensitive and the fluorescent signal can decrease at higher temperatures. Temperature fluctuations between samples, or between samples and standards, can cause problems.
Make sure that the buffer and Qubit reagent in DMSO are at room temperature. The buffer and Qubit reagent should be stored at room temperature, not in the refrigerator. Even after 2-3 hours at room temperature, buffer previously stored at 4°C can remain below room temperature.
Make sure your samples and working solution are not too warm (including those straight from a centrifuge). Samples kept in the Qubit instrument too long or read multiple times can warm up. If you want to perform multiple readings of a single tube, you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading. Also, do not hold tubes in your hand for very long before reading them in the instrument, since this can warm the sample, resulting in a low reading.
3.Ensure that you have prepared the Qubit working solution correctly (1:200 dilution using the buffer provided in the kit). Ensure that you have prepared the standard tubes correctly (10 µL of each standard in 190 µL of the working solution). Ensure that the tubes are filled with at least 200 µL (both standards and samples).
4.Ensure that the reagents and standards you are using are less than 6 months old, and that the standards have been stored correctly. The Qubit reagent stock solution should be protected from light as much as possible.
5.Ensure that you have selected the correct assay on the Qubit Fluorometer for the Qubit assay you are performing.
6.Ensure that the lid is completely closed when reading standards and samples.
7.Use recommended tubes (both so the tube does not obstruct the lid, and for optical clarity). Some types of tubes can have high autofluorescence that will affect the assay.
8.Did you enter the number of microliters of stock you pipetted into the working solution into the Qubit instrument? If so, the reading after giving the Qubit Fluorometer this information is the concentration of your stock solution. If you did not, the reading you got is for the concentration in the assay tube (the tube you put into the Qubit Fluorometer) and not your stock solution.
9.If you are comparing Qubit assay results to concentration obtained by UV absorbance, and the concentration based on absorbance is significantly higher, it may be because of nucleic acid or protein contamination. The Qubit assays are much more specific for DNA, RNA, or protein than absorbance readings.
Please see our suggestions below:
PicoGreen dye and other fluorescence-based quantification reagents are not recommended for quantifying dye-conjugated nucleic acids. The attached dye molecules can interfere with either binding and/or fluorescence output of the quantification reagents.
Strands that are roughly in the 20-mer range or shorter show a lower level of signal. For dsDNA samples that are composed of mostly short strands, the reagent may still be used, but one should use a dsDNA standard that is of comparable length as the sample.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
The Qubit Fluorometer contains highly optimized algorithms that calculate the concentration of the sample using either the Qubit assays or the Quant-iT DNA assays. The Quant-iT PicoGreen DNA assay may be adapted to the Qubit Fluorometer using the MyQubit firmware. The performance of all of these assays is similar.
The Quant-iT PicoGreen DNA assay is the most established assay and the most general-purpose (http://tools.thermofisher.com/content/sfs/manuals/PicoGreen-dsDNA-protocol.pdf). It requires the dilution of the standard DNA and buffer but can be adapted for use with either cuvettes, microplates, or the NanoDrop 3300.
The Quant-iT DNA assays provide a ready-to-use buffer and pre-diluted standard DNA for analyzing a large number of samples (>20 samples) using a 96-well microplate with no further adaptation.
The Qubit assays (https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-assays/myqubit.html) are intended for low throughput (<20 samples), and are only used on the Qubit Fluorometer.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
The exact excitation/emission wavelength information is proprietary. Here are the approximate excitation/emission wavelengths:
- Qubit dsDNA HS Assay: ~500 nm/ ~530 nm
- Qubit dsDNA BR Assay: ~510 nm/ ~530 nm
- Qubit ssDNA Assay: ~490 nm/ ~520 nm
- Qubit RNA HS Assay: ~640 nm/ ~670 nm
- Qubit RNA BR Assay: ~640 nm/ ~670 nm
- Qubit microRNA Assay: ~500 nm/ ~520 nm
- Qubit Protein Assay: ~470 nm/ ~570 nm
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
Yes, you can, for Qubit instruments developed after the original Qubit (1.0) Fluorometer. See MyQubit assay instructions here (http://www.thermofisher.com/us/en/home/life-science/laboratory-instruments/fluorometers/qubit/qubit-assays/myqubit.html.html).
Generally, the cleaner the sample the better. Some salts, proteins, and detergents are tolerated in the assays; see the specific assay protocol for which ones and at what concentrations.
The accuracy and sensitivity of the Qubit quantitation assays are the same as that of a microplate reader. This was a requirement during product development. The detection limits for each Qubit kit can be found on the corresponding product manual, which can be found by searching our website by keyword or catalog number.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
No. The Qubit DNA and RNA kits only quantify the amount of either DNA or RNA in the sample. The Qubit fluorometer cannot take absorbance readings to provide a A260/A280 ratio or detect protein in nucleic acid samples. This can be done with the NanoDrop instrument. If your sample contains protein or other contaminants that can affect the assay, it should be further purified.
If your sample may contain both DNA and RNA, one may use either (or both) the DNA and RNA Qubit kits and compare with samples treated with either RNase or DNase to get an accurate determination of DNA or RNA, respectively.
All Quant-iT and Qubit kits are compatible with all fluorometers and microplate readers that have the appropriate light sources and filters. You won't have access to the algorithm in the Qubit fluorometer for generating the standard curve provided by the instrument, instead, you must make a few dilutions of the highest standard DNA or RNA (Standard #2) in the Qubit kits to generate a standard curve with multiple data points.
No, we do not recommend this. Some of the dyes in the original Quant-iT kits (those NOT listed as “for use with the Qubit fluorometer”) are not compatible with the Qubit Fluorometer. In addition, the new Quant-iT kits (labeled as “for use with the Qubit Fluorometer”) have standards formulated to be compatible with the Qubit fluorometer internal algorithms for the respective assays. The Qubit Fluorometer-compatible kits are also less expensive per assay if you are processing fewer than 20 samples at a time.
The Qubit 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay Kits (Cat. Nos. Q33230, Q33231, Q33265, Q33366) are newer and have a simplified workflow compared to the original Qubit dsDNA Quantification Assay Kits (Cat. Nos. Q32850, Q32851, Q32853, Q32854). The original Qubit kits contain separate dyes and buffer components that must be mixed together to make a working solution, in which the dye degrades after a few hours. With the newer Qubit 1X dsDNA Assay kits, the dye is premixed with a buffer that keeps the dye stable long-term and can be added directly to DNA samples.
You can find more information about the Qubit 1X dsDNA assay, by clicking on the Technical Note provided below:
Qubit 1X dsDNA assays: simplified workflow and improved performance
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.
Detection of somatic variants with the Oncomine Myeloid Research Assay GX has been verified down to 5% allele frequency.
Here is a list of kits that are compatible with Oncomine Myeloid Research Assay GX:
Nucleic acid isolation - RNA samples
- MagMAX mirVana Total RNA Isolation Kit, Cat. No. A27828
- PureLink Total RNA Blood Kit, Cat. No. K156001
- RNaseZap RNase Decontamination Solution, Cat. No. AM9780, AM9782, AM9784
Nucleic acid isolation - DNA samples
- MagMAX DNA Multi-Sample Ultra 2.0 Kit, Cat. No. A36570
- PureLink Genomic DNA Mini Kit, Cat. No. K1820-01
The recommended minimum sample input is 27.75 ng of DNA (1.11 ng/µL minimum concentration) and 14.25 ng of RNA (0.95 ng/µL minimum concentration).
The recommended controls are:
DNA Controls:
- Seraseq® Myeloid Mutation DNA Mix, Cat. No. 0710-0408 (SeraCare®)
- Myeloid DNA Reference Standard, Cat. No. HD829 (Horizon™)
- AcroMetrix™ Oncology Hotspot Control, Cat. No. 969056
RNA Controls:
- Universal Human Reference RNA, Cat. No. 740000 (Agilent™)
- Seraseq® Myeloid Fusion RNA Mix, Cat. No. 0710-0407 (SeraCare®)
Multiplex sequencing of up to 8 Oncomine Myeloid Research Assay GX samples (8 DNA+ 8 RNA) per lane on a GX5 Chip can be performed in a single run. There are 4 lanes on the GX5 Chip, so 32 total samples (32 DNA+ 32 RNA) can be run on one GX5 Chip (8 samples per run).
The Oncomine Myeloid Research Assay GX (Cat. No. A47857) includes the 2-pool DNA panel, the 1-pool RNA panel, Genexus Strip 1, and Genexus Strip 2-AS. The DNA panel is provided in two packs of 8 tubes (4 tubes of Myeloid DNA Pool 1 and 4 tubes of Myeloid DNA Pool 2 per pack). The RNA panel is provided in one pack of 8 tubes (Myeloid RNA Pool 1). Each primer pool in the panel is provided in pairs of tubes, where each tube pair contains one tube with primers in position 1 and one empty uncapped tube in position 2. Three 8-strip packs of the Genexus Strip 1 and Genexus Strip 2-AS (24 total of each strip) are provided with each kit.
The contents of each Oncomine Myeloid Research Assay GX kit are sufficient for up to 32 samples (32 x 3‑pool reactions: 32 x 2‑pool DNA reactions and 32 x 1‑pool RNA reactions).
The Oncomine Myeloid Research Assay GX includes 3 pools of Ion AmpliSeq oligonucleotide primers (a 2-pool DNA panel and a 1-pool RNA panel).
The Oncomine Myeloid Research Assay GX targets key genes and fusions associated with major myeloid disorders, including acute myeloid leukemia (AML), myeloid dysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). The DNA panel comprises 40 key genes while the RNA panel includes a broad fusion panel of 29 driver genes, enabling detection over 600 unique fusion isotypes. See flyer for gene targets (https://assets.thermofisher.com/TFS-Assets/CSD/Flyers/Oncomine_myeloid_assay_gx_flyer.pdf).
The Oncomine Myeloid Research Assay GX is a comprehensive targeted next-generation sequencing (NGS) assay designed for sensitive detection of myeloid disorder-associated DNA mutations and fusion transcripts in blood and bone marrow samples.
When stored as directed, the Qubit dsDNA HS Assay Kit is guaranteed stable for 6 months from the date of receipt.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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
If you are not using the Ion Library Equalizer Kit for library normalization, one of the following can be used (but the library should be at 100 pM):
- Ion Library TaqMan Quantitation Kit
- Qubit 3.0 Fluorometer or Qubit 2.0 Fluorometer
- Qubit dsDNA HS Assay Kit
- Agilent 2100 Bioanalyzer Instrument
- Agilent High Sensitivity DNA Kit
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
We recommend two methods for gDNA quantification:
- Qubit 3.0 Fluorometer (Cat. No. Q33216) with Qubit dsDNA HS Assay Kit (Cat. Nos. Q32851, Q32854)
- qPCR using TaqMan RNase P Detection Reagents Kit (Cat. No. 4316831)