The specificity was significantly reduced to 15~30% even with the addition of the primer blockers and increased human cot-1 DNA in the hybridization mix (data not shown).
Preparations of cDNA were diluted (1 25 to 1 100) and tested by qPCR using RBCS1-specific primer pairs (Table 1) designed using Primer Express software (Applied Biosystems), which were preliminarily tested for their specificity and efficiency against a cDNA mix (data not shown).
There were no statistical differences (Kruskal Wallis p = 0.94) found in the distribution of the patient groups across the three clinic years in terms of both the patient population mix (i.e., predefined patient groups) and the patient population visit mix (data not shown).
Baseline and demographic characteristics did not differ substantially by year of diagnosis, although individual years had some small differences in the Charlson score (slightly declined over the study period), and the distributions of age, region, education levels, and treatment mix (data not shown).
The intensity of the actin cortices was found to be higher at lower temperatures, most likely due to the temperature-induced changes in the balance between actin assembly and disassembly dynamics in our system, also exemplified by the increased lengths of actin comet tails at 20°C compared to 30°C in the same motility mix (data not shown).
If field data show that there is no evidence of any geographical trend for a given parameter, then mixing data from different geographic regions may not be problematic.
I am not sure if it is a good idea to mix data from all lineages to study the correlation.
The sequences showed a patient-specific clustering with high bootstrap support (>90%), which argues against the occurrence of contamination and sample mix-up (data not shown).
Phylogenetic analysis of V3 sequences from the entire PCR products showed distinct clusters of sequences for each patient that excluded any possibility of sample contamination or mix-up (data not shown).
Similar results were observed if the HIV-1 vector was incubated for only one hour with the A3-CCR5 cells before washing and mixing with PM-1 (data not shown).
