The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

Abstract

Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99%) samples, no significant difference was detected between the median log 10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99%) samples, no significant difference was found in median log 10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of realtime PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

MYCN protein stability is a better prognostic indicator in neuroblastoma

“…Since no reliable MYCN antibody is used in IHC, clinicians and researchers usually detect MYCN amplification status at the nucleic acid level. Conventional polymerase chain reaction (PCR) [ 7 ], quantitative real-time PCR (qPCR) [ 8 , 9 ], semi-quantitative differential PCR (SQ-PCR) [ 10 ], droplet digital PCR (ddPCR) [ 11 ], FISH [ 12 ], chromogenic in situ hybridization (CISH) [ 13 ], and multiplex ligation-dependent probe amplification (MLPA) [ 14 ] are some common methods. The FISH result is an important index of risk stratification [ 15 ].…”

Semi-quantitative RT-PCR analysis of transcripts encoding protease inhibitor inHevea brasiliensisMuell. Arg latex

“…The main reason for its popular use is that the semi-quantitative method provides a combination of traditional PCR and transcript quantification at a relatively low cost [22]. The semi-quantitative PCR was equally confirmed to generate results as well as the more advanced methods [24].…”

MYCN Fish combined with immunohistochemistry is a more prognostic factor for neuroblastoma

“…Current guidelines for the assignment of risk group used by the International Neuroblastoma Risk Group (INRG) and the Children' Oncology Group (COG) heavily weigh MYCN ampli cation. Till now, for no reliable MYCN antibody, a number of methodologies used in clinical and laboratory tests are con ned to nucleic acid level, such as conventional polymerase chain reaction (PCR) (Ma et al 2016) , quantitative real-time PCR(q RT-PCR) (Feng et al 2007, Kojima et al 2013, semi-quantitative differential PCR (SQ-PCR) (Souza, Souza, Sanabani, Giorgi and Bendit et al 2009), droplet digital PCR (ddPCR) (Somasundaram et al 2019), uorescence in situ hybridization (FISH) (Somasundaram et al 2019) chromogenic in situ hybridization(CISH) (Bhargava, Oppenheimer, Gerald, Jhanwar and Chen et al 2005), multiplex ligation-dependent probe ampli cation (MLPA) (Vícha and Eckschlager et al 2008)and so on. MYCN gene status tested by FISH is integrated into the risk classi cation presently (Pinto et al 2015).…”