Comparison of the normalized relative expression levels of housekeeping genes (HKGs) between the three subgroups.

<p>The relative expression levels of remaining seven genes were normalized against the Normalization Factor based on the geometric mean of the expression level of the best-performing HKGs (<i>B2M</i> and <i>RPLP0</i>). Data are presented as mean ± SE. ^a<i>P</i><0.05.</p

Flow cytometry analysis on CD4⁺ T cells in lymphocyte suspension and in purified CD4⁺ cells by immunomagnetic depletion with the human CD4⁺ T Cell Isolation Kit II.

<p>Cells were directly stained with conjugated fluorescently labeled antibodies for CD4 (BD Biosciences) in lymphocyte suspension and in purified CD4⁺ samples.</p

Determination of the housekeeping gene expression stability by NormFinder.

<p>The stability value is estimated using the model-based approach. Having considered both the intra- and inter-group variation, a lower stability value represents a smaller systematic error that would be introduced when using the studied gene.</p

Data of candidate housekeeping genes (n = 28).

<p>GM, geometric mean; AM, arithmetic mean; Min, minimal value; Max, maximal value; CV, coefficient of variance.</p

Correlation analysis of candidate housekeeping genes (HKGs) versus BestKeeper index.

<p>^aAll 8 candidate HKGs showed strong correlation (correlation coefficient <i>r</i> value from 0.69 to 0.93) and were combined into an index, which was then used to compute the correlation between each HKG and the index.</p

Housekeeping genes evaluated in the present study.

<p>Housekeeping genes evaluated in the present study.</p

Ranking the housekeeping genes (HKGs) according to their expression stability M determined using geNorm.

<p>A stepwise exclusion of the least stable HKG was conducted to obtain the mean expression stability value M of remaining HKGs until the two most stable HKGs were identified. The genes are ranked according to M values.</p

Primer sequences for housekeeping genes.

<p>Primer sequences for housekeeping genes.</p

Demographic characteristics of the participants.

a<p>mean ± SD;</p>b<p>median (range);</p>c<p><i>P</i><0.05;</p>d<p><i>P</i> = 0.073; BMI, body mass index; FEV₁, forced expiratory volume in 1 s; FVC, forced vital capacity; ACT, Asthma control test; GINA, Global initiative for asthma; AQLQ, Asthma quality of life questionnaire; Ig, immunoglobulin; PD₂₀FEV₁, provocative dose of methacholine causing a 20% drop in FEV₁; ICS, inhaled corticosteroid; LABA, Long-acting β₂-agonists.</p