Mireasy mini kit

According to the manufacturer's manufacturer, total RNA, including small RNAs, was isolated and purified from the buffy coat of patients using miReasy Mini Kit (Cat #217004; Qiagen, Hilden, Germany) instruction for cells samples. The RNA concentration and purity were detected using a QIAexpert spectrophotometer (Cat #1038703; Qiagen). Then complementary DNA (cDNA) was synthesized from 10 ng of total RNA using a TaqMan MicroRNA Reverse Transcription Kit (Cat #4366597; Applied Biosystem Lithuania) with a specific RT primer (Cat #4440887 hsa-miR21-5p; Cat #4427975 U6 snRNA; Applied Biosystem, Lithuania) according to the manufacturer's protocol. miRNA expression was measured with a Rotor-Gene Q (Cat #R0515102; Qiagen) using PrimeTime Gene Expression Master Mix (Cat #1055772; IDT) and the TM primer (Cat #4440887 hsa-miR21-5p; Cat #4427975 U6 snRNA Applied Biosystem). The two-step PCR condition was 3 m at 95°C, 45 cycles with 5 s at 95°C, and 30 s at 60°C (10 μl reaction volume and 2 μl cDNA template). All samples were run in triplicate. A threshold cycle (C_t) value was obtained for each amplification cycle, and ΔC_t was calculated as the C_t difference between target miRNA and U6. miRNA expression was calculated using the 2^−ΔΔCt method.

RNA was extracted by using a miReasy mini kit (Qiagen) following the manufacturer’s instructions, as detailed previously (19 (link)). A DNase digestion step was performed to ensure no genetic DNA contamination. RNA (1 μg) was used to synthesize complementary DNA by using oligo-dT-adaptor primers and Moloney murine leukemia virus reverse transcriptase (Promega). Gene expression was determined by using custom-designed primers (Sigma) and SYBR Green reagents (Applied Biosystems). Primer sequences are presented in Table 1. Quantification of gene expression was performed with the use of a Step One Plus reverse transcriptase–polymerase chain reaction machine (Applied Biosystems). Equal efficiency of the reverse transcription of RNA from all groups was confirmed through quantification of expression of the housekeeping gene β-actin (Actb). The expression of Actb did not differ between groups (effect of maternal diet, P = 0.9; effect of CoQ₁₀ supplementation, P = 0.8; control: 153 ± 32; recuperated: 144 ± 24; control CoQ₁₀: 143 ± 12; and recuperated CoQ₁₀: 157 ± 21 average copy numbers).

Small RNAs were isolated and enriched from longissimus muscle biopsies using a miReasy Mini kit and an RNeasy MinElute Cleanup kit (Qiagen) according to manufacturer’s protocols. The quality and quantity of small RNA were assessed with an Agilent 2100 Bioanalyzer (Agilent) using an Agilent small RNA kit.