Dr. Accola: When we want to sequence a sample, we obtain three FFPE tissue slides, one of which is stained with H&E (Hematoxylin and Eosin). Macro-dissection is the first step. We put an H&E slide next to an unstained slide and circle the area from which we want to extract DNA. We scrape that area off the unstained slide and put it into the tube with mineral oil, which is the beginning of the Maxwell® CSC extraction. What we’ve learned is that as long as there is sufficient tumor and tissue, we only need one slide for the Maxwell® CSC for DNA extraction. Once extracted, we quantify the sample for NGS. For sample quantitation we use the Quantus™ Fluorometer, which is more accurate at the lower end than absorbance (when quantitating small amouts of DNA). The Life Tech fluorometer is the Qubit™, though we find that the Quantus™ Fluorometer works equally well (Figure 2). The cost is also a little bit less per sample with the QuantiFluor® dye. Specifically we have been using the QuantiFluor® ONE dsDNA System. We like it because the pre-diluted format takes one step away from the assay setup, so now it’s one fewer step than running the Qubit™ assay. Ultimately, you need to have a very good quantitation estimate before you start the library prep—you’re targeting a normalization input of 10ng. This is where less is probably better than more, because if you find that your library prep is unsuccessful the last thing you want to do is use more DNA in the reaction. So, quantification before library preparation is critical. Read the Complete Article » Comparison of FFPE Quantitation Methods. 250 200 150 100 50 0 050 100 150 200 Promega Quantus™ (ng/µL) 250 60 50 R2 =0.8583 40 30 10 20 0 010203060 40 Promega Quantus™ (ng/µL) Figure 2. DNA was purified from 12 FFPE tissue sections using the Maxwell® CSC Instrument and Maxwell® CSC DNA FFPE Kit and quantitated using the NanoDrop® (UV-absorbance) instrument, Qubit™ fluorometer and Quantus™ fluorometer. The NanoDrop® quantitation method overestimated DNA, while the Qubit™ and Quantus™ methods performed similarly. enews | March 2016 50 R2 =0.9662 NanoDrop (ng/µL) Life Tech Qubit (ng/µL) Pagina 16
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