Quantitative Molecular Reference Standards

Do You Monitor the Limit of Detection of Your Molecular Assays?
Verify the true sensitivity of your molecular standards every day

Genomic DNA Reference Standards

Horizon Diagnostics’ (HDx) Quantitative Molecular Reference Standards are available primarily in two formats, as Formalin-Fixed, Paraffin-Embedded (FFPE) cells, and as genomic DNA (gDNA).

Genomic DNA is an ideal tool to establish the limit of detection of molecular assays, but to be effective it must be extracted from cell lines with precisely controlled copy numbers and allelic frequencies, which is not easily achievable with patient derived cell lines. Horizon Diagnostics’ reference standards therefore represent an ideal solution, due to the highly characterized, reproducible nature of the cell lines used to generate the gDNA. 

Horizon Diagnostics’ gDNA Reference Standards are derived from engineered mutant (50% mutant) and matched “normal” (0% mutant) cell lines. Mutant cell lines are all heterozygotes, and in the majority of cases this means there is a single mutant allele and a single normal allele. Where the copy number is 2N, a 100% mutant gDNA sample indicates that 100% of the cells the gDNA was extracted from contained the mutation, however as only one of the two copies of the gene from that cell will actually contain the mutation, the sample will actually have an allelic frequency of 50% for that mutation.  Genotypes with a copy number of 3N and with a single mutant allele would have an allelic frequency of 33%. Mutant and “normal” gDNA can be precisely blended to generate samples with specific allelic frequencies.

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Operator Variability
Pre-Analytical
Analytical
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Sample FFPE DNA Extraction DNA Quantitfication Platform Sanger QPCR NGS Molecular Assay Output

Sources of variability within a standard molecular assay workflow

Products

  • HDx provides a range of pre-validated reference standards at allelic frequencies including but not limited to 1%, 5%, 10%, 20% and 50%.  The range of frequencies allows end users to generate standard curves where each data point is verified by digital PCR, thereby negating the requirement to perform dilutions manually.
  • Routine Validation Packs provide gDNA at a sufficient range of allelic frequencies with matched wild-type to enable users to generate a standard curve, with each dilution previously validated by Horizon using digital PCR.  Packs are currently available for BRAF, EGFR and KRAS gene variants.

All Genomic DNA Reference Standards are provided at 5ng/µl concentration and in multiples of 100 ng tubes unless otherwise stated.  Custom aliquoting is available upon request.

Key Features

  • Allelic frequency can be modified as required
  • Precisely validated for copy number and allelic frequency using Droplet Digital PCR
  • Use of matched mutant and normal gDNA replicates typical tumor genetics
  • Horizon Diagnostics’ Reference Standards are a highly consistent, renewable resource

Applications

  • Routine monitoring of analytical performance, for both specificity and sensitivity
  • Standard curve generation to establish limit of detection
  • Independent assay verification
  • Assay/ platform optimization
  • Staff training

Sample


Tumor profiles and genotype can often be specific to individual samples and depending on the sample the proportion of normal versus tumor cells can be highly variable. Genoytping a sample from a 10% tumor cell population may challenge the limit of detection of the molecular assay compared to a sample from a 100% tumor cell population.

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Operator Variability


Successful execution of the analytical phase is completely dependent on correct performance during the pre-analytical phase

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FFPE


Sample fixation processes are highly variable and will impact on the quality of the extractable DNA. High-grade formalin for a short time (i.e. less than 24 hours) will preserve the quality DNA within the samples and provide a greater DNA yield compared to Low-grade formalin for a longer time (i.e. more than 24 hours).

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DNA Extraction


Isolation of genomic DNA from formalin fixed paraffin embedded (FFPE) tissues is a critical step for molecular diagnostic (MDx) assays. It is essential that the maximum amount of DNA is recovered from the FFPE tissues and its quality is sufficient to perform the necessary MDx assays. 

The quality and quantity of DNA can vary widely depending on the extraction method/kit used.

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DNA Quantitfication


Success or failure in DNA analysis applications often comes down to whether or not the appropriate amount of nucleic acid is used in the analysis and if the quality of that nucleic acid is sufficient. UV absorbance is one of the most popular methods for quantitating nucleic acids, however, there are many methods available, each with its own strengths and weaknesses. 

At DNA concentrations below 10ng/µl UV absorbance is often inaccurate up to a factor of 3 - 5 fold. 

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Platform


The limit of detection of a molecular diagnostic assay can vary from 1% to 25%. This is dependent on the assay design as well as the platform choice. Not all platforms are capable of distinguishing between subtle nucleic acid changes.

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Sanger Sequencing


Sanger Sequencing has a limit of detection cutoff between 15% and 25% and this sensitivity depends on the gene, mutation and assay design. 

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QPCR


A quantitative polymerase chain reaction (qPCR) will have a limit of deteciton of between 1% and 10%. This sensitivity will be defined by the assay design and specificity of the probe(s).

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NGS


Next generation sequencing (NGS) is offering a way to quickly and affordably establish baseline tumor genetics and the limit of detection is between 1% and 5%.

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Molecular Assay Output


The format of the molecular assays output will be different depending on the platform and software used. 

Analysis and interpretation of sample data is critical for effective tumor profiliing. This is becoming more difficult with multiplex NGS platforms and our reference standards can be used to assist with data interpretation and benchmarking.

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