Includes all controls.

EntroGen’s DNA Fragmentation Quantification assay (FQA) predicts sample integrity and concentration for downstream real-time PCR and next generation sequencing (NGS) assays.

DNA fragmentation, presence of PCR inhibitors, and chemical modifications due to formalin-fixed paraffin embedding (FFPE) affect sample integrity and compromise the sensitivity and reliability of downstream real-time qPCR and NGS assays. For example, fragmented DNA leads to a lower conversion rate of input DNA to adaptor-ligated library during library preparation in NGS workflows, resulting in low sample diversity. This necessitates more library amplification cycles prior to sequencing and leads to high levels of duplication and low depths of coverage, therefore increasing the risk of false-positives and false-negatives. Instruments based on spectrophotometric and fluorometric analysis provide the total DNA concentration but cannot distinguish between fragmented and unfragmented (amplifiable) DNA or detect PCR inhibitors Therefore, sample inputs based on concentrations determined by these methods (i.e. Nanodrop, Qubit) may lead to inconclusive or inaccurate results in qPCR and NGS.

In order to utilize patient samples effectively and control the quality of qPCR and NGS data, the DNA Fragmentation Quantification assay (cat no. FQA-RT40) provides two metrics to guide loading optimal DNA input for any downstream qPCR and NGS assay. The FQA assay uses three amplicon sizes (37bp, 150bp, 301bp) of a reference gene and 5 standards to calculate the absolute copy number of amplifiable DNA and determine sample integrity through the fragmentation ratio (F-ratio).

The kit comes with 5 DNA standards that set the standard curve used for determining the copy number of amplifiable DNA. After determining the copy numbers for each fragment size two F-ratios-150bp/ 37bp and 301bp/37bp- are calculated. The F-ratios are indicative of sample quality and help predict the optimal amount of input sample for downstream assays. Automated data interpretation that provides the absolute copy number of amplifiable DNA and F-ratios is available for FQA.

Workflow

First, isolate DNA using the extraction kit of your choice. After DNA isolation, set up reactions using reagents provided in the DNA Fragmentation Quantification kit and run the assay on a compatible real-time PCR instrument. The assay requires  less than 2 hours to complete.  Automated analysis is available to assist with data interpretation.

Equipment and Materials

DNA Fragmentation Quantification Assay requires a real-time PCR instrument.

The kit includes:

• Reaction mix
• Primer-probe mixes (specific for each amplicon size)
• DNA standards

The kit does not include reagents and materials for DNA isolation.

The reagents in the FQA kit are sufficient for 64 tests including all controls and samples.

USA:  DNA Fragmentation Quantification Assay (FQA) is provided for research use only (RUO). Not for use in diagnostic procedures.

Europe:  DNA Fragmentation Quantification Assay (FQA) is provided for research (RUO) and diagnostic (CE-IVD) purposes.