Rotor gene q 5plex hrm instrument

To a 200 μl PCR tube, 39 μl of water, 5 μl of 10 × ThermolPol Reaction Buffer, 2 μl of probe (10 pmol) and 1 μl of target ssDNA (5 pmol) were added and mixed well. The solution was heated to 85°C and then gradually cooled down to 37°C. Then 3 μl of λ exo (1.67 U) was added and the detection was performed at 37°C on a Rotor-Gene Q 5plex HRM Instrument (QIAGEN, Hilden, Germany) with gain level of 8. Fluorescence intensity was measured once a cycle (5 s per cycle) for 240 cycles. The excitation and emission wavelengths were set to 470 and 510 nm, respectively. The rate of fluorescence increase was determined by the slope of the linear portion of the time curve.

To a 200 μl PCR tube, 39 μl of water, 5 μl of 10 × ThermolPol Reaction Buffer, 2 μl of probe (10 pmol) and 1 μl of the complementary strand (5 pmol) were added and mixed well. The solution was heated to 85°C and then gradually cooled down to 37°C. Then, 3 μl of λ exo (NEB) was added, and the detection was performed at 37°C on a Rotor-Gene Q 5plex HRM Instrument (QIAGEN, Hilden, Germany). The amount of λ exo as trimer was 0.53 pmol (2.5 U, final concentration 10.6 nM) for Figures 1A and 3, Supplementary Figure S2, S3, S4 and S6A, 0.36 pmol (1.7 U, final concentration 7.2 nM) for Figure 2, Supplementary Figure S5 and S6B, and 0.18 pmol (0.83 U, final concentration 3.6 nM) for Figure 5, Supplementary Figure S7 and S8B. Fluorescence intensity was measured once per cycle (5 s per cycle). The excitation and emission wavelengths were set at 470 and 510 nm, respectively, for the probes labeled with FAM and 585 and 610 nm, respectively, for the probes labeled with ROX. The rate of fluorescence increase was determined by the slope of the linear portion of the time curve. All experiments were repeated at least three times.

For EGFR mutation detection, the Rotor-Gene Q 5plex HRM instrument was used with the therascreen EGFR RGQ PCR kit (therascreen EGFR assay; Qiagen, Inc.). This assay is approved for use in the United States, Europe, Japan, and China, and the kit is based on the amplification-refractory mutation system (ARMS) and Scorpion PCR technology, which enable the sensitive and selective site-specific detection of 29 types of somatic mutations in EGFR (24 (link)). The reaction conditions were as follows: 95°C for 15 min for 1 cycle; 95°C for 30 sec and 60°C for 60 sec for 40 cycles. The analysis was performed using the Rotor-Gene Q series software, version 2.0.2 (Qiagen, Inc.). For comparison between the three types of cytological samples (cell pellets, cell-free supernatants and FFPE cell blocks) using cell lines, the cycle quantification (Cq) value of the mutant allele in each sample was compared using the therascreen EGFR ‘Deletions’ assay, which detects EGFR E746_A750del. For the validation study comparing BLF specimens to FFPE tissue specimens, the manufacturer-supplied cut-off delta Cq (ΔCq) values were used to determine the result whether positive or negative for the mutation in each EGFR mutation reaction.