Since we started with viral RNA, we increased the background threshold for variant calling to 0.5%, what we believe is the biologically relevant frequency threshold.
However, the fluorescence intensity was not higher than the background threshold for any of these probes, indicating that mRNA concentrations of these cytokines (including IFN-γ) in infected and uninfected mucosal scrapings were too low to detect by microarray analysis, probably due to the relatively low percentage of cytokine-producing (immune) cells present in intestinal mucosa [ 6].
A cell designated as positive for KORp-ir if optical density was above a standardized background threshold value for each treatment group, as determined by using Metamorph software (Dovingtown, PA).
Then, for each strand and each position i it is checked whether the coverage c i crosses the background threshold T. For each position that satisfies this criterion it is checked whether the possible transcript can be extended downstream, with each subsequent position x satisfying the condition c i + x > T.
For the identification of operons, it was examined if a gene and the next downstream gene in sense direction are connected by a number of paired mappings exceeding the background threshold T for this data set.
The background threshold was set the same for each image analyzed.
For a gene to be considered as detected, the normalized gene counts for a specific transcript had to be significantly (Student's t test, p < 0.05) above background threshold count value for the negative controls.
Then, a binary value was declared for each miRNA in each sample as expressed/unexpressed, based on the results of the miRNA microarray (background threshold was variable and estimated manually for each sample).
In any given sample type, transcripts were detected for 10,067 to 17,070 genes (hybridization signal above background threshold); transcripts were detected for 21,939 different genes considering all of the tissues, i.e. 92% of the P. glauca genes represented on the microarray.
The line scan tool provided in the LSM software (Zeiss) was used to scan several areas of sections that were not stained with the appropriate antibodies to determine the background thresholds used for the co-localization tool in the calculation of the relative area of K5K8DP TECs.
