Trizol reagent extraction method

Isolation of total RNA: Total RNA was isolated from cardiac tissues by standard TRIzol^® Reagent extraction method (Thermo-Fisher Scientific, Bremen, Germany). The extracted RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water by passing solution several times through a pipette tip. Aliquots were used immediately for reverse transcription (RT), otherwise stored at −80 °C
Reverse transcription (RT) reaction: The complete Poly(A)⁺ RNA isolated from cardiac tissues was reverse transcribed into cDNA in a total volume of 20 µL using RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas, Dreieich, Germany). The obtained cDNA was used for DNA amplification through real-time polymerase chain reaction (RT-PCR) [40 ].
Real time-Polymerase Chain Reaction (RT-PCR): StepOne™ real-time PCR System from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine rat’s heart cDNA copy number. PCR reactions were set up using SYBR^® Premix Ex TaqTM (TaKaRa, Biotech. Co. Ltd., Shiga, Japan). The sequences of specific primers of the RAR-α gene used are listed in Table 4. At the end of each qPCR, a melting curve analysis was performed at 95.0 °C to check the quality of the used primers. The relative quantification of the target to the reference was determined by using the 2^−ΔΔCT method [41 (link)].

Total RNA was extracted from harvested cells or human tissues by a Trizol reagent extraction method (Thermo Fisher Scientific). To determine the expression of miR-200c, U6 expression was used as an endogenous reference. To determine the NDN expression, 18s was used as an internal control. The oligo primers were shown as follows: 5′-GGTAATACTCCCGGGTAAT-3’ (forward) and 5’-CAGTGCGTGTCGTGGAGT-3’ (reverse) for miR-200c, 5’-CGAGCTCATGTGGTACGT-3’ (forward) and 5’-CGATGACATCTTTCACCATGTC-3’ (reverse) for NDN. The data were normalized to those of the controls and analyzed by the 2^–ΔΔCt method [27 (link)].