Establishing a reference dataset for the authentication of spinal muscular atrophy cell lines using STR profiling and digital PCR

Abstract

Fibroblasts and lymphoblastoid cell lines (LCLs) derived from individuals with spinal muscular atrophy (SMA) have been and continue to be essential for translational SMA research. Authentication of cell lines helps ensure reproducibility and rigor in biomedical research. This quality control measure identifies mislabeling or cross-contamination of cell lines and prevents misinterpretation of data. Unfortunately, authentication of SMA cell lines used in various studies has not been possible because of a lack of a reference. In this study, we provide said reference so that SMA cell lines can be subsequently authenticated. We use short tandem repeat (STR) profiling and digital PCR (dPCR), which quantifies SMN1 and SMN2 copy numbers, to generate molecular identity codes for fibroblasts and LCLs that are commonly used in SMA research. Using these molecular identity codes, we clarify the familial relationships within a set of fibroblasts commonly used in SMA research. This study presents the first cell line reference set for the SMA research community and demonstrates its usefulness for re-identification and authentication of lines commonly used as in vitro models for future studies.

Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

“…6B) and total SMN ( Fig. 6C) copy number measurements of the blinded clinical samples were concordant with those results obtained using QuantStudio 3D array dPCR 27,31 , including a sample (MND12) with 5 copies of total SMN. Bland Altman analysis of the results from SMN1 (Fig.…”

Infants Diagnosed with Spinal Muscular Atrophy and 4 SMN2 Copies through Newborn Screening – Opportunity or Burden?

“…The methodological problem with the secure determination of the number of SMN2 copies has to be addressed. A recent study shows the discrepancy between standard qPCR and the more accurate ddPCR [50,51]. In this pilot project, the problem was circumvented by carrying out confirmation diagnostics via MLPA from whole blood (multiple determination in case of uncertainties), and independent confirmation of the result by a second laboratory.…”

Short tandem repeat profiling for the authentication of cancer stem‐like cells

“…To protect the identity of the subjects, eight core STR loci plus Amelogenin were used to report on the identity of a given sample. The following core loci were recommended, D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX and CSF1PO 20,21 . The information about the other loci can be shared in strict confidence to researchers and biorepositories 22,23 .…”

“…The following core loci were recommended, D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX and CSF1PO. 20,21 The information about the other loci can be shared in strict confidence to researchers and biorepositories. 22,23 Tables 1 and 2 show STR profiles of CTSCs and GSCs, respectively.…”