This method, termed multiplex-ready PCR, combines the advantages of the M13-tailed primer method and multiplex PCR in a single step, closed tube assay.
High-resolution melting (HRM) of small amplicons in the presence of intercalating dyes is increasingly used as a means to reveal sequence variation [ 5, 6], and has the advantage of being a closed-tube assay.
After a short centrifugation, the Repli-g reaction was added on top of the solidified AmpliWax®, the tube closed and the assay performed in a real-time Mx3005P-cycler (Stratagene).
Although amplicons for HRM analysis are shorter than that used in mismatch cleavage assay, HRM is a closed-tube, low cost and fast assay; no digestion and gel separation steps are required.
A closed-tube RET genotyping assay using a saturating DNA dye, unlabeled probes, and amplicon high-resolution melting analysis was developed.
The Clark electrode chamber was closed and the assay was started simultaneously with the recording of the third baseline data point in the XF24.
A closer analysis of the assays and their performance reveal the difficulty in using any of these methods as a confirmatory approach, owing to the secondary induction of necrosis in apoptotic cells.
Statistical analysis of ELISA, IFA and Western blotting titers (from all volunteers and time-points measured) revealed a close correlation between all assays.
The era of steroid analysis via immunoassay is drawing to a close as these unspecific assays are replaced by high-throughput, specific, sensitive mass spectrometry (MS) analyses (5).
Therefore, we believe that the use of genuine human NC cells for the MINC assay closes an important gap in test batteries assessing the many aspects of developmental toxicity.
