Advanced mirna cdna synthesis kit

Representative liver tissue was stored in RNAlater stabilization solution (Ambion, Austin, TX, USA) at − 80 °C. miRNAs were extracted using the miRVana miRNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer's instructions. The integrity and amount of the RNA fraction highly enriched in sRNA species ≤ 200 nt was determined using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA purity was assessed from the ratio of A260/A280; where 1.8–2.1 is expected^{34 (link)}. Ten ng of sRNAs were used to performed retrotranscription using the Advanced miRNA cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's instructions. Reverse transcription products were diluted to 50 µL, and 2.5 µL of the diluted sample were used for qPCR reactions, with a total volume of 10 µL/Rx and 1X Thermo specific TaqMan probes were used for qPCR for mmu-miR-34a-5p, mmu-miR-122-5p, mmu-miR-21a-5p and mmu-miR-103-3p, in a LigthCycler 96 equipment (Roche Molecular Systems, Pleasanton, CA, USA). qPCR was performed as follows: 1 cycle at 95 °C for 20 s, 40–60 cycles 95 °C for 1 s followed by 60 °C for 20 s. Probes catalog number are shown in Supplemental Table 1. Gene expression was normalized against mmu-miR-16-5p. Analysis was performed using the 2^−ΔCT method^{35 (link)}.

The expressions of miR-197 and silent information regulator 1 (SIRT1) in H9c2 cells were determined. Concretely, TRIzol reagent (15596026, Thermo Fisher, USA) or miRNeasy Mini Kit (217084, Qiagen, Germany) was used to extract total RNA from the cells following the operational manual. Next, the complementary DNA (cDNA) was synthesized using the Reverse Transcription Kit (4368814, Thermo Fisher, USA) or the Advanced miRNA cDNA Synthesis Kit (A28007, Thermo Fisher, USA). Subsequently, qRT-PCR was carried out. In a nutshell, the synthetics were reacted with PrimeScript RT Master Mix (RR036A-1, TAKARA, Japan) or TaqMan Fast Advanced Master Mix (A44360, Thermo Fisher, USA), and then developed in a Real-Time PCR system (7300, Applied Biosystems, USA). The expression levels of genes were quantified using the 2^−ΔΔCT approach [22 (link)]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were applied as the internal controls. The sequences of forward (F) and reverse (R) primers were listed as follows: miR-197 (F: 5′-ATTACTTTGCCCATATTCATTTTGA-3′; R: 5′-ATTCTAGAGGCCGAGGCGGCCGACATGT-3′); SIRT1 (F: 5′-TTAAAGCCGTGAGCCTCCAG-3′; R: 5′-ACAAAAAGCATTCCATACCGTCA-3′); GAPDH (F: 5′-AGTTAATGCCGCCCCTTACC-3′; R: 5′-CAGGGCTGACTACAAACCCA -3′); U6 (F: 5′-CGATCCAATCGGAACGGGAT-3′; R: 5′-AGGCGCCATTTCCCAACATA-3′).

RNA was isolated from PBMC lysates using the Quick RNA MiniPrep Isolation Plus Kit (ZymoResearch, Freiburg, Germany). A miRNeasy serum spike-in miR-39 miRNA (Qiagen, Hilden, Germany) was integrated in the micro-RNA isolation procedure. Equal amounts of RNA (10ng) were reverse transcribed using the Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific). cDNA samples were diluted 1:60, followed by qPCR using the miRCURY LNA SYBR Green PCR Kit (Qiagen). Primers used for the detection of Hsa-142-3p/5p (YP00204291/YP00204722) were purchased from Qiagen. The samples were processed in duplicates on a StepOnePlus Cycler (Thermo Fisher Scientific). Data were normalized by miR-39 and related to healthy control donor miR. Thus, results were expressed as the x-fold difference compared to the healthy control.