3.3. The M/N ratio in NB tumor tissue and plasma
The total DNA was extracted from 96 cases NB plasma samples and 60 cases NB clinical samples of NB tumor tissues, and the MYCN copy number in plasma and tissues. The OD260/OD280 of total DNA extracted from plasma and tissue specimens were detected with a range from 1.80 to 1.90, which indicated that the extracted DNA were in good quality (Table S2). First, the MYCN amplification status of these 96 cases of NB tumor tissues was detected by FISH, and the 96 cases of NB were subsequently divided into an MNA group (n = 25) and non-MNA group (n = 71) according to MNA status. There were no statistically significant differences between the MNA group and the non-MNA group in terms of gender, age, and stage of diagnosis (Table 1). Following, the M/N ratio of corresponding plasma (n = 96) was detected by single-tube duplex RT-PCR approach. The experimental results showed that the M/N values in the plasma of the MNA group (n = 25) were all greater than 2 whereas the M/N values in the plasma of the non-MNA group (n = 71) were all less than 2 (Figure 3A and Table 1). The plasma M/N ratio in the MNA group (n = 25, median M/N ratio = 4.90) was significantly higher than that in the non-MNA group (n = 71, median M/N ratio = 1.22), P < 0.001 (Figure S2A). Meanwhile, the M/N ratio in 60 cases of NB tumor tissues, including 16 MNA cases and 44 non-MAN cases, was detected by single-tube duplex RT-PCR approach. The experimental results also showed that the M/N ratios in MNA group (n = 16) were greater than 2 and the M/N ratios in non-MNA group (n = 16) were less than 2 (Figure 3B and Table 1). The M/N ratios in the tissue of the MNA group (n = 16, median M/N ratio = 2.94) were significantly higher than that of the non-MNA group (n = 71, median M/N ratio = 1.44), P < 0.05 (Figure S2B). When the M/N ratio cut-off value was set to 2, the detection results of the 96 cases of plasma and 60 tumor tissues based on the detection method established in this study were completely consistent with the detection results of FISH. Lastly, the M/N ratio in 60 cases of NB with both plasma and tissue samples was detected by the developed single-tube duplex RT-PCR approach. Experimental results showed that the correlation coefficient between plasma M/N ratio and tumor tissue M/N ratio was 0.9496 (Figure 3C). Therefore, the M/N ratios in NB plasma and tumor tissues are consistent with each other.
Table 1 Characteristics of patients according to plasma and tissueMYCN status