Includes all controls.
Internal Quality Control Assay (IQCA) predicts sample quality and concentration for downstream EntroGen qPCR assays.
Optimal sample quality is critical for the detection of mutations using real-time PCR. Formalin-fixed paraffin-embedded (FFPE) tissue is commonly used as a source of DNA for real-time PCR and provides valuable insight into patient-specific disease. However, DNA samples extracted from FFPE tissues are prone to fragmentation, various chemical modifications, and may contain PCR inhibitors that can compromise PCR amplification efficiency. In addition to the quality of the sample, loading the optimal concentration of DNA for each qPCR assay is also crucial, especially when a patient sample has limited availability. Instruments based on spectrophotometric and fluorometric analysis provide the total DNA concentration but not the amplifiable DNA concentration. In other words, instruments such as Nanodrop and Qubit cannot detect PCR inhibitors or distinguish between fragmented and amplifiable DNA, the latter being the template in downstream qPCR assays. Therefore, sample inputs based on concentrations determined by Nanodrop and Qubit can easily underload or overload the PCR reaction and affect the reaction’s efficiency, quality of the results, and necessitate a re-run of the sample.
The Internal Quality Control Assay (IQCA) is designed to save time, samples, and reagents by measuring the amount of amplifiable DNA and detecting the presence of PCR inhibitors in a sample prior to running any downstream EntroGen qPCR assay. IQCA amplifies an internal control gene and predicts the amount of DNA input based on its Ct values. IQCA requires an input of 1µL of sample DNA (adjusted to 10 ng/µL) to the provided reaction mix. After running the assay on a compatible real-time PCR instrument, the results can be imported into an automated analysis spreadsheet that predicts the volume of sample input necessary for optimal downstream qPCR performance.
After DNA isolation, set up reactions using reagents provided in the IQCA kit and run the assay on a compatible real-time PCR instrument. The assay may be completed in approximately 1 hour and 15 minutes from assay set up to results. Automated analysis is available to assist with data interpretation for certain instruments (please contact firstname.lastname@example.org for more information).
Equipment and Materials
The IQCA kit requires a real-time PCR instrument capable of detecting the VIC fluorophore.
The kit includes:
- 2x Reaction Master Mix
- Primer-probe mix for the detection of the internal control gene
The kit does not include reagents and materials for DNA isolation.
The reagents in the IQCA kit are sufficient for 50 reactions.