137 A novel liquid nitrogen-free snap freezer Table 1. RNA integrity of cell line samples processed with different freezing methods. Aluminum vials with lysates of K562 suspension cell line were alternatingly snap-frozen in the snap freezer or in liquid nitrogen. Three samples were left at room temperature for two hours before freezing in liquid nitrogen as a positive control sample. RIN, RNA integrity numbers. Freezing method RIN value Replicate 1 Replicate 2 Replicate 3 Liquid nitrogen 9,40 9,50 9,30 Snap freezer 9,10 9,30 9,20 +2hr Control sample 9,40 9,20 9,30 Normal liver tissue biopsies Characteristics and analysis details of five consecutive patients who underwent liver surgery are presented in Supplementary Table 1. For patient 01 only 3 normal liver tissue biopsies were available (phosphoproteomics) and for patients 02, -03- and -04, 6 biopsies per patient could be evaluated for phosphoproteomics, RNA integrity analysis and RNA sequencing. These biopsies were snap-frozen alternatingly using the three freezing methods (Figure 1B). In total, twelve 14G core needle biopsies from four patients were processed for global phosphoproteomics, with a median protein input of 500 µg per sample. A total of 15,262 unique peptides were identified, of which 10,395 (68%) were phosphorylated. The median number of phosphopeptides per sample was 6,742 (range 5535 – 7601). A total of 9,966 phosphosites were identified (86% serine, 13% threonine and 1% tyrosine), with a median of 5794 (range 4710 – 6573) per sample. Unsupervised clustering of the phosphoproteome revealed clear separation of replicates from the four patients (Figure 3A). Subclustering of snap freezer- and LN2-frozen samples, separate from the +2hr controls, was observed in 2 of 4 patients. RNA isolation was successfully performed in tissues from 2 of 3 last mentioned patients. An additional set of nine liver biopsies was obtained from a fifth patient (05, Supplementary Table 1). RNA quality was insufficient in one of the biopsies, leaving 11 samples for downstream analysis. There were no significant differences in RIN values between the samples processed using the 2 freezing methods (p = 0.89 by t-test). Samples that were left at room temperature for 2 hours before immersion in LN2 had RIN values comparable to the other two freezing conditions, indicating that RNA is a stable molecule, even after a prolonged cold ischemia time (Supplementary Table 1). After RNA sequencing, unsupervised cluster analysis of gene expression profiles showed a clear separation of the samples from individual patients (Figure 3B). 5
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