Droplet digital pcr

The cell lines were sequenced by Sanger Sequencing and SMN1/SMN2 copy number was determined by Dr. Matthew Butchbach, Nemours Biomedical Research and their Biomolecular Core Lab. Briefly, cell lines were tested for SMA by genomic PCR (ddPCR) and Sanger sequencing [10 (link)]. Cells were analyzed for single nucleotide polymorphisms (SNP) within SMN2 by Sanger sequencing and showed no variant SNPs [38 (link),39 (link)]. SMN1 and SMN2 copy number was measured using digital droplet PCR (Bio-Rad) according to manufacturer`s instructions [10 (link)].

Patients with unresectable pancreatic carcinoma undergoing FOLFIRINOX chemotherapy were enroled under the research protocol CCR3085 (SSGCC) that has received approval from the Research Ethics Committee, London. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Baseline whole blood was collected in EDTA-treated tubes; cells were removed by centrifugation for 15 min at 1,500 × g using a refrigerated centrifuge (4 °C). The supernatant was collected and centrifuged for further 10 min at 1,500 × g at 4 °C. The resulting supernatant was designated plasma and was transferred into clean RNAse free tubes and stored at −80 °C. RNA was extracted from 200 uL using the miRCURY RNA isolation kit (Exiqon, Qiagen, UK) following supplier instructions. Fixed starting volume was maintained for each sample. Digital droplet PCR (BioRad, Berkeley, CA, USA) was performed as previously described^{47 (link)} using the MIR1307 TaqMan probe (Life Technologies, Paisley UK). A no-template control and a negative control for each reverse transcription reaction were included in every assay and at least 10,000 droplets were assessed for each sample. MiR-1307 expression was assessed blinded to clinical data. In the Kaplan Meier analysis, patients were divided into two groups: low MIR1307 and high MIR1307 according to the median value.

In CRC cases, DNA was extracted and purified from formalin-fixed, paraffin-embedded tumor tissue collected during routine clinical practice at the Department of Clinical Pathology, Umeå University Hospital, Umeå, Sweden. KRAS was analyzed by sequencing the activating mutations in codon 12 and 13 using Big Dye v. 3.1, according to the manufacture protocol (Applied Biosystems, Life Technologies, Foster City, CA, USA) [23 (link)]. The mutational status of BRAF^V600E was analyzed using TaqMan allelic discrimination assay (reagents from Applied Biosystems) and digital droplet PCR (reagents from Bio-Rad Laboratories, Hercules, CA, USA) [24 (link)]. CIMP status was determined using MethyLight real-time PCR and an 8-gene panel as previously described in [25 (link)]. Cases were classified as CIMP-negative if there was no promotor methylation in any of the eight genes defined as a percentage of methylated reference (PMR) value above 10%; CIMP-low if there was promotor methylation in one to five genes; and CIMP-high if there was promotor methylation in six to eight genes. MSI status was assessed by immunohistochemical analysis of MLH1, MSH2, MSH6, and PMS2 [25 (link)]. Samples lacking tumor cells with nuclear staining for any of the mismatch repair proteins were categorized as MSI.

Basal serum tryptase and total IgE (tIgE) were quantified by the FEIA ImmunoCAP (Phadia Thermo Fisher Scientific, Uppsala, Sweden). In addition, sIgE to wasp (i3), recombinant (r) Ves v 1 and rVes v 5 were determined in patients with WVA. Ratios for sIgE-to-tIgE were calculated.
KIT D816V mutation in PB and BM was determined, as described earlier (24 (link)), using digital droplet PCR (Bio-Rad, Hercules, USA). Quantification of the KIT D816V allele burden in the BM cells was available in 14 patients with a CMCD with wasp venom anaphylaxis and 13 patients with a CMCD patients without anaphylaxis with a sensitivity of at least 0.01% positive alleles.

We randomly selected a subset of all high-quality exonic de novo SNVs, all de novo indels and all CSVs for validation in probands and available parents. We used Primer3^{69 (link)} to design primers to span at least 100 bp upstream and downstream of a putative variant, avoiding regions of known SNPs, repetitive elements, and segmental duplications. DNA from whole blood, if available, was used to amplify candidate regions by polymerase chain reaction and to assay with Sanger Sequencing. For CNVs, we validated all high-confidence de novo exonic and all clinically significant CNVs in whole blood DNA (if available) of probands and available parents using TaqMan^TM Copy Number Assay (Applied Biosystems), SYBR® Green qPCR (Thermofisher) or digital droplet PCR (BioRad). Experimental validation rates were 94.8%, 85.7%, and 87.5%, respectively, for de novo SNVs, indels, and CNVs (Supplementary Data 3 and 4).