Mircury lna universal rt microrna pcr

Serum samples were pooled together for each mouse group (n = 10). RNA extraction was performed with the mirVanaTM PARISTM kit (Thermo Fisher). RNA was eluted in 50 μL RNase-free water, concentrated in 10 μL after NaOAc precipitation, and quantified by using a Nanodrop spectrophotometer (ND8000 Labtech, Wilmington, DE, USA). Quantification of miRNAs was performed using Exiqon miRCURY LNA Universal RT microRNA PCR. Total RNA (20 ng) was converted into poly(A) primed universal cDNA, and microRNAs were quantified in duplicate for each sample with miRNA-specific LNA primers on the LightCycler480 (Roche) following the manufacturer’s guidelines (miR-133a, miR-133b, miR-1, miR-206, and miR-378). Quantification cycle (Cq) values were calculated with the LightCycler 480 SW 1.5.1 using the 2nd derivative max method. qRT-PCR results, expressed as raw Cq, were normalized to the miRNAs identified as the most stable, miR-16, miR-142-3p, and miR-223. The relative expression was calculated using the 2^−ΔΔCt method.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from all the 3 cell lines with or without treatment with 5-Aza-dc (5 µM) at room temperature for 4 days and human tissues. Universal cDNA Synthesis kit (Exiqon; Qiagen, Inc., Valencia, CA, USA) was used to synthesize first-strand complementary DNA. miRCURY LNA™ Universal RT microRNA PCR (Exiqon; Qiagen, Inc.) was used to conduct qPCR determining miRNA. cDNA synthesis was conducted at 95°C for 12 min, and the qPCR was conducted with the following thermocycling conditions: 97°C for 5 min, followed by 35 cycles at 95°C for 30 sec, 65°C for 30 sec and 73°C for 1 min, and a final step at 73°C for 10 min; samples were then kept at 4°C until use. U6 was used as an endogenous control. Primers of hsa-miR-193a-3p (product no. 204591) were obtained from Exiqon. The forward primer of miR-193a-3p was 5′-CTGAGGGCTGGGTCTTTGC-3′ and the reverse primer was 5′-GCCGAGAACTGGGACTTTGT-3′. The forward primer and reverse primer of U6 were 5′-CTCGCTTCGGCAGCACA-3′ and 5′-ACGCTTCACGAATTTGCGT-3′, respectively.