Total RNA was extracted using a protocol combining the Oragene®•RNA Self-Collection Kit (DNA Genotek) and the mirVanaTM miRNA isolation kit (Ambion, Austin, TX). In brief, 300 µl of whole saliva mixed with 300 µl of Oragene®•RNA was incubated for 1 hour at 50ºC, then heated at 90ºC for 15 minutes and allowed to cool to room temperature. Afterwards, 48 µl of the Oragene®•RNA Neutralizer solution was added. Samples were mixed, incubated on ice for 10 minutes, and then centrifuged at 10 000 x g for 3 minutes at room temperature. The supernatant was collected and the isolation of total RNA was completed using the mirVanaTM miRNA isolation kit with modifications to the manufacturer’s protocol. First, for the lysis step, the addition of the Lysis/Binding buffer and the miRNA Homogenate Additive were bypassed and instead an equal volume of acid-phenol:chloroform was added directly to the collected supernatant. Second, only 50 µl of 95ºC elution solution was used to elute RNA. RNA was quantified using a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). An Agilent 2100 Bioanalyzer (Santa Clara, CA) was used to detect the size distribution of total RNA, as well as determine the quality of the RNA.
Label
FAM
Label protocol
n/a
Hybridization protocol
n/a
Scan protocol
n/a
Data processing
The analysis of expression of the >700 miRNAs was performed by the DNA Core at the Interdisciplinary Center for Biotechnology Research Center at the University of Florida, according to the manufacturer’s protocol except the pre-amplification step was omitted. The NormFinder algorithm was used to identify the optimal normalization of miRNA among the 25 most abundantly expressed miRNAs detected.
