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Quality Control

DNA and RNA concentration

NanoDrop is used to measure concentration and purity of nucleic acids before starting a microarray experiment. The concentration of nucleic acid is dependent on what array to be used. OD260/280 should be close to 2.0 for RNA dissolved in water and 1.8 for DNA. OD260/230 should preferably be above 2. A lower ratio might indicate  contamination, such as GITC or phenol residues from extraction. A low OD260/230 reading can also occur as an artefact in samples with low concentration (< 50 ng/µl).

RNA quality

Agilent 2100 Bioanalyzer is used to analyze the integrity of nucleic acids. Total RNA samples can be processed on either a Nano chip, Pico chip or a Small RNA chip, depending on the sample amounts and concentrations. The RNA Integrity Number (RIN) software algorithm allows the classification of total RNA, based on a numbering system from 1 to 10, with 1 being the most degraded and 10 being the most intact. Total RNA for expression studies needs to be of high and even quality. Useful information about QC measurements using the NanoDrop and the Bioanalyzer can be found here.

FFPE DNA concentration and quality

Qubit and Rotor Gene are used for checking concentration and quality of FFPE DNA prior to the Illumina Methylation assay. Qubit can be added as a QC, but qPCR on the RotorGene is standard. The Illumina HD FFPE QC kit is used on the RotorGene to determine weather the DNA samples are candidates for the Illumina FFPE Methylation assay or not. A ”delta Ct” value is calculated and should be lower then 5 to pass.