QIAamp ccfDNA/RNA Kit

The QIAamp ccfDNA/RNA Kit enables co-purification of cell-free DNA and RNA, including vesicular and non-vesicular nucleic acids. Starting from 1–4 ml plasma, the protocol uses midi spin columns, removing the need for a vacuum step. For nucleic acid elution, MinElute spin columns allow for small elution volumes of 14 µl. The kit ensures high yields of cell-free DNA and total RNA including miRNA from plasma and serum samples.

The QIAamp ccfDNA/RNA protocol is specially designed to allow both DNA and RNA elution from 1–4 ml plasma or serum (see figure QIAamp ccfDNA/RNA Kit procedure). Yields of cell-free DNA and RNA are comparable to or better than the QIAamp Circulating Nucleic Acid Kit (see figure High recovery of ccfDNA and RNA compared to the QIAamp Circulating Nucleic Acid Kit). Unlike other kits, the QIAamp ccfDNA/RNA Kit ensures high yields of RNA and DNA (see figure Competitive performance without compromising on DNA or RNA yields). The QIAamp ccfDNA/RNA Kit further achieves higher RNA integrity than other kits, making it broadly suited for any downstream application (see figure High integrity of RNA).

The QIAamp ccfDNA/RNA Kit provides an efficient way to isolate both DNA and RNA from the same plasma or serum sample. The procedure uses midi spin columns, allowing vacuum-free processing of 1–4 ml sample input volume (see figure Scalable yields of cfDNA and RNA between 1–4 ml plasma sample amount). Small elution volumes are achieved using MinElute columns. The complete protocol is phenol-free so that it can be carried out without working under a fume hood. The protocol and reagents have been optimized to ensure high yields of DNA and RNA.

The QIAamp ccfDNA/RNA Kit protocol consists of initial cell-free DNA/RNA isolation, followed by cleanup and concentration. The cleanup steps can be automated on the QIAcube.

Cell-free DNA/RNA isolation

Serum or plasma (1–4 ml) are mixed with Buffer RPL to lyse exosomes and release RNA. The sample is then mixed with Buffer RPP and centrifuged to precipitate proteins. Isopropanol is added to the supernatant to provide the appropriate conditions for nucleic acids to bind to the silica membrane. The sample is then applied to the midi spin column, where the DNA and RNA molecules bind to the membrane. The column is washed twice with different buffers before the cell-free DNA and RNA is eluted simultaneously.

Cleanup

Lysis buffer and ethanol are added to the eluate before transfer to an RNeasy MinElute column. The column is subsequently washed with wash buffer and ethanol before the cell-free DNA and RNA is eluted.

For any liquid biopsy sample that requires parallel analysis of DNA and RNA mutations or for samples with low mutant allele frequencies, the QIAamp ccfDNA/RNA Kit efficiently isolates complete DNA and RNA, including miRNA, from any plasma or serum sample.