QIAamp DNA FFPE Tissue Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

Purification of DNA and RNA from FF and FFPE tissue
To identify extraction methods resulting in optimal nucleic acid quality and yield, RNA and DNA were purified from the FFPE blocks of the ( and ) using commercially available kits as described below. Up to six 10 µm sections were cut from each tumour block and placed in sterile 1.5 mL centrifuge tubes ready for extraction. Tubes containing cut FFPE sections for RNA purification were stored at −80°C until use. DNA and RNA purifications from the matching FF liver and tonsil samples were done using a QiaSymphony robot in combination with the QIAsymphony DSP DNA Mini Kit and QIAsymphony RNA Mini Kit (both from QIAGEN). DNA was purified from FFPE samples using QIAamp DNA FFPE Tissue (QIAGEN) and Nucleospin FFPE DNA (Machery-Nagel) kits; RNA was purified from FFPE samples using miRNeasy FFPE (QIAGEN), Nucleospin FFPE RNA (Machery-Nagel) and ExpressArt FFPE RNAready (Amp Tec) kits. In order to test parallel purification of RNA and DNA from the same FFPE sections, the Nucleospin FFPE RNA/DNA kit (Machery-Nagel) and RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion) were included. Finally, in order to test automated purification procedures, DNA and RNA were purified on a QiaSymphony robot using specialized FFPE programs and the QIAsymphony DSP DNA Mini Kit and QIAsymphony RNA Mini Kit (QIAGEN), respectively. All extractions were performed in triplicate, each on three FFPE sections. Because of a limiting amount of material, extraction from breast samples with Recover All was done in duplicates only. Purifications were performed following the manufacturers' protocols with the following modification: Deparaffinisation before DNA and RNA extraction using the QIAamp FFPE DNA and ExpressArt FFPE RNAready kit, respectively, was done using QIAGEN's Deparaffinisation Solution instead of the xylene included in the kits. All DNA samples were RNase treated and all RNA samples were DNase treated during the purification. Following purification, all samples were washed on UF filter plates and eluted in QIAGEN's Elution Buffer (EB, [10 mM Tris-HCl, pH 8.5]). The fragmentation profiles of the purified RNA and DNA were estimated by on-chip electrophoresis of a 1 µL sample on an Agilent 2100 Bioanalyzer (Agilent Technologies). Since only double-stranded (ds) DNA is active in TruSeq library formation, both the total absorbance at 260 nM (NanoDropND-1000 Spectrophotometer) as well as a dsDNA specific assay (Qubit dsDNA BR Assay Kit/Qubit 2.0 Fluorometer, Invitrogen) was used to quantify the DNA concentration. The RNA concentration was determined using only the total absorbance at 260 nM.

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Manufacturer protocol

Download the product protocol from Qiagen for QIAamp DNA FFPE Tissue Kit below.

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