Fig 1.
Effect of freezing in the CXCE-buffer extraction.
Comparison of Cq values without freezing (not frozen, yellow) and with freezing (frozen, blue) in the TREC (A) and the SMN1 (B) assays.
Fig 2.
MagNA pure 96 vs. CXCE-buffer extraction.
Comparison of TREC amplification signals from MagNA Pure 96 extraction (black amplification curves) vs. CXCE-buffer extraction (red amplification curves).
Fig 3.
Evaluation of pre-analytic DNA carry-over in TREC (A) and SMN1 (D) amplification curves. In total 364 samples, including 40 white/empty samples, 80 TREC negative samples (dark blue lines: white/empty punches; light blue dashed lines: TREC negative punches; orange lines: positive samples (for TREC≈300 000 copies/ml)). Boxplots and the defined cut-off values based on the Cq values of the TREC (B) and SMN1 (C) assays with TREC and SMN1 positive samples in orange, negative samples in blue. Further 110 samples showed no amplification for TREC and 8 no amplification for SMN1 and therefore cannot be included in the Boxplot. (D) represents 364 samples, including 40 white/empty (blue lines) and SMN1 positive samples (orange lines).
Table 1.
Contaminations using the CXCE-buffer extraction.
Results of empty punches embedded between TREC positive samples using the CXCE-buffer extraction and qPCR detection system (Total N = 120).
Table 2.
Contaminations using the EnLite system.
Results of empty punches embedded between TREC positive samples using the endpoint PCR-based EnLite system (Total N = 766).
Fig 4.
Comparison of amplification curves (A) of the middle/inner DBS area (M) vs. the periphery/edge DBS area (P) with the CXCE-buffer extraction. The performance of the amplification curves for the nucleic acids of TRECs shows the impact of the chosen punching area within a DBS of the same sample (blue lines/boxes: middle DBS Area (M), orange lines/boxes: periphery DBS area (P)). Direct comparison of the respective Cq values for areas M and P for TREC (B) and SMN1 (C) DNA.