Log Likelihood Ratio comparison for human samples.
For human samples, after RNA isolation (Maxwell® 16 LEV simplyRNA Tissue Kit on Maxwell 16 Instrument, Promega, Mannheim, Germany) RNA was transcribed into cDNA (High Capacity cDNA Reverse Transcription Kit, Life Technologies) according to standard protocols.
By using a conservative approximation of 0.15 IU/ml as a cutoff point, the new ELISA-based technique demonstrated a sensitivity of 100% and a specificity of 95% for human samples and for experimental animal samples.
For human samples the clinically tested anti migraine compounds were applied to the isolated arteries that that had been precontracted with 30 mM K+.
Primers to ribosomal protein S16 (for mouse samples) and RPL13 (for human samples) were used to normalize cDNA loading.
GAPDH for human samples and rer1 for mouse samples have been additionally tested, in several cases, to confirm the results.
Colonic epithelial cells were isolated from mice using a protocol modified from one previously described for human samples [56].
Program setting for human samples were as follows: 10 min at 95°C, 40 cycles of 15 sec at 95°C 20 sec at 63°C.
For human samples, a tonsil specimen was used as calibrator, whereas a gastric sample from one of the naïve mice was used as calibrator in mouse assays.
For human samples, the informed written consent have been obtained from the immediate families of three victims of car accidents (please see details below).
Negative controls were compared with the human samples in both datasets, and the numbers of negative controls that yielded positive results for human samples, i.e. contaminated blanks, were then compared between Linköping and Stockholm/Uppsala.
