diagnosis: bronchopulmonary dysplasia (BPD) tissue: tracheal aspirate mild/moderate bpd: no severe bpd: yes
Treatment protocol
Tracheal aspirates were obtained from premature patients with bronchopulmonary dysplasia. Total RNA was purified from TAs and the expression of 1066 human miRNAs was detected with a PCR array.
Extracted molecule
total RNA
Extraction protocol
MicroRNAs were purified from 0.5 mL of TAs with the Plasma/Serum RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada).
Label
SYBR Green
Label protocol
Small RNAs were retro-transcribed from 200 ng of total RNA using the miScript II RT kit (Qiagen). The expression of 1066 human miRNAs was assayed with the Human miRNome miScript® miRNA PCR Array (QIAGEN, MIHS-3216Z).
Hybridization protocol
n/a
Scan protocol
n/a
Description
severe BPD sample name in data files: 12
Data processing
Data were analyzed in excel using Ct values for each sample, normalized to global means, and fold changes were calculated using the 2-ΔΔCT method. Matrix normalized template shows miRNA Ct values normalized to global mean ΔCt = (Ct_miRNA − Ct_global mean) For fold change calculations, ΔΔCt based fold-change values were obtained using an average of control sample Ct values (2^-ΔΔCt, where -ΔΔCt = -[ΔCtsample -ΔCtavgcontrol])). A fold change of 25 was considered as cutoff. Matrix non-normalized are raw Ct values. Matrix normalized are normalized to the global mean. Fold changes are calculated with the livak method (2^-ΔΔCt, where -ΔΔCt = -[ΔCttest -ΔCtcontrol]). Sample 1 was used as control for most miRNAs, except when the expression was below the detection limit, in which case we used other samples (2, 3, 4, 5, 20).
