Spiked methlyated DNA and non-target poly-A RNA were purified from 4 ml plasma using the QIAamp Circulating Nucleic Acid Kit, with an elution volume of 45 μl. Bisulfite conversion was carried out using the EpiTect Bisulfite Kit (QIAGEN), and methylation-specific PCR (MSP) was performed using real-time MSP assays and components of the QuantiTect Multiplex PCR Kit on the ABI PRISM 7900HT Sequence Detection System. Data from two fully methylated loci were analyzed.
DNA fragments (200 bp and 1000 bp) in equal amounts were added to 24 plasma samples. DNA was purified from 5 ml plasma using the QIAamp Circulating Nucleic Acid Kit, with an elution volume of 80 μl. DNA yield was quantified by duplex, real-time PCR of 66 bp targets specific for each DNA fragment using the QuantiTect Multiplex PCR Kit.
Circulating DNA was purified from 5 ml plasma using the QIAamp Circulating Nucleic Acid Kit or from 1 ml plasma using the QIAamp MinElute Virus Vacuum Kit, with elution volumes of 100 μl. DNA yield was quantified by duplex, real-time PCR of a 66 bp amplicon and a 500 bp amplicon within the 18S rRNA gene using the QuantTect Multiplex PCR Kit. The difference between amplification of the 500 bp amplicon and 66 bp amplicon shows that the DNA is fragmented, resulting in a lower abundance of intact 500 bp target sequences compared with the shorter 66 bp target. (Note that the difference is greater than the fivefold difference in sample input volumes.)
Circulating RNA was purified from 4 ml pooled plasma using the QIAamp Circulating Nucleic Acid Kit, with an elution volume of 50 μl. The standard kit protocol was compared with the specialized protocol for miRNA. miRNA 30b yield was quantified using a TaqMan microRNA assay (Applied Biosystems), and miRNA 16 yield was quantified using a Human miScript Assay (QIAGEN). The lower CT values with the miRNA protocol indicate higher yields of the miRNA species.