RNA extraction and MicroRNA analysis
Out of 44 participants, 22 participants from the active treatment group,
and 21 subjects from the placebo group were randomized and evaluated
regarding the microRNA. The microRNAs were analyzed at the study start
and at the end of study in both the intervention and the placebo groups.
The vials were retained on ice before chilled centrifugation at 3000g
and then frozen at -70°C, and no sample was melted more than twice.
Total RNA was extracted from 100μL of serum sample using RNX-Plus
reagent (Cinnagen, Iran) followed by DNase I digestion (Takara Bio Inc,
Shiga, Japan) according to the user manual. The concentration and purity
of extracted RNAs was evaluated by Nano drop ND-1000 spectrophotometer
(Nano drop Technologies, Wilmington, DE, USA) reading at wave length
230, 260 and 280 nm. The RNA samples were liquated in RNase free tubes
and stored at -70°C before use.
Complementary DNA (cDNA) was synthesized by Prime Script™ RT reagent kit
(Takara Bio Inc, Shiga, Japan). Based on manufacturer protocol.
Real-time PCR was performed using the SYBR® Premix Ex Taq™ II (Takara)
and relative gene expression was calculated as 2−ΔΔCt.
The list of primers for specific genes has been illustrated in Table4.
The miRNA Snordu6 was used as an internal control.