The cKIT D816V Reference Standard is FFPE derived from a highly characterized homozygous diploid HCT116 cell line, genetically modified with advanced genome editing technology to include the mutation D816V in cKIT on both chromosomes. The variant allele frequency (VAF) is 100%, and can be used for quality control in the FFPE DNA extraction process.
This reference standard can be used to assess the performance of your NGS, sanger sequencing and qPCR assay, including the limit of detection, accuracy, and precision.
| Catalog number | RM208 |
| Format | FFPE |
| Fixation | 10% neutral buffered formalin solution |
| Quantity | 1 scroll |
| Cell density | > 500,000 cells per scroll |
| Volume | 20 µm section |
| DNA Yield | > 400 ng per scroll |
| Cell line | HCT116 |
| Storage | 4 ºC |
| Shelf life | 3 years from the date of manufacture |
| Quality Control | H&E staining Agarose gel electrophoresis Qubit 4.0 fluorometer Sanger sequencing ISO 9001:2015 certified |
Single-gene reference standards are designed to detect somatic mutations when validating Sanger sequencing and PCR assays. These reference standard mutations are oncology-based focused for genetic testing assays and are available in gDNA, RNA and FFPE formats.
GeneCopoeia offers highly-characterized, biologically-relevant homozygous OncoSpotTM Reference Standards that provide consistent and reliable resources. These standards can be incorporated into your workflow to optimize protocols by evaluating assay sensitivity, specificity and accuracy.
Advantages of Reference Standards:
- Optimize your DNA isolation protocols prior to testing patient samples
- Monitor the impact of workflow changes on downstream results
- Establish limits of detection
- Accurately evaluate batch-to-batch variability during your quality control process
- Ideal for diagnostic assay development.
GeneCopoeia utilizes genome editing technology to enable precision genome editing and expansion from a single clone. This technology eliminates plasmid DNA integration which allows for better primer design and effectiveness. The mutant and wild type sequence alignments for both the mutant and wild type cell lines can be found on the product datasheet/website.
All Reference Standards are prepared and analyzed using our ISO9001 certified quality control process. The cell lines used for manufacturing Reference Standards also carry endogenous mutations, and these mutations may also be called during analysis. More information on endogenous mutations may be found on the product website.We assess the gDNA by agarose gel electrophoresis by spectrophotometry (A260/A280). Each reference standard lot is checked for quality control and that each test result is within acceptance criteria before being released.
We assess the quantity of gDNA by Qubit assay.
Our DNA Reference Standards are manufactured by extracting genomic DNA from single clone human cell lines. Reference Standards are available as either mutant or wild-type at a defined locus, and the mutation status is confirmed by Sanger sequencing. We offer 50ng/µl of gDNA Reference Standards.
The cell lines are mildly-formalin fixed and then homogenously embedded in paraffin wax to generate the FFPE Reference Standards. We prepare 20 µm sections using 5.0 mm diameter FFPE cell blocks.
Yield greatly depends on FFPE extraction method used. Internally we extract gDNA from FFPE by using the QIAamp DNA FFPE tissue extraction kit and achieve yields of ≥ 400ng per FFPE curl. These kits, can achieve extraction yields of 200ng – 500ng per FFPE sample. It is important to follow the gDNA isolation protocol rather than the total nucleic acid isolation protocol.
If you observe no yield or less than 50 ng total from isolation protocol, it is likely that the cell pellet was lost during the deparaffinization stage. It is important to use the original tube during the xylene treatment. If you observe yields of 50 – 200 ng per FFPE section, it is likely that during the elution step that the entire DNA was not filtered through the column. It may be beneficial to perform a two 50 μl elution compared to a single 100μl elution to yield higher DNA totals.
To generate an LOD curve, 1 vial of mutant DNA and 1 vial of matched wild type DNA is required. Depending on which application the DNA Reference Standards are used for, the LOD dilution curve allelic frequencies may vary.
- Standard LOD by Application:
- Sanger sequencing 15-25%
- qPCR 1%
- NGS below 1%