Mircury rna isolation kit tissue

Total RNA was isolated from the tumor and non-cancerous tissue samples using the miRCURY RNA Isolation Kit-Tissue (Exiqon, MA, USA) and diluted to a final concentration of 5ng/μl for cDNA synthesis. The Universal cDNA Synthesis Kit II (Exiqon, MA, USA) was used for cDNA synthesis according to the manufacturer’s protocol. Expression levels of the miR-134 in the tumor and normal tissue samples were analyzed by Quantitative Real Time PCR (qRT-PCR) using the LightCycler 480 (Roche Diagnostics, Mannheim, Germany). The U6 small nuclear RNA was used as a reference gene for normalization. PCR reactions were performed in a final volume of 10 μl containing 1XSYBR Green Master Mix, 54 ng of each miR-134 or U6 gene-specific primers and cDNA. The reaction conditions included a polymerase activation/denaturation of 10 minutes at 95°C, 40 cycles of amplification at 95°C for 10 seconds, 60°C for 1 minute, followed by melting curve analysis, and a cooling step of 30 seconds at 40°C. Relative miRNA levels were calculated using the 2^−ΔΔCt method [26 (link)].

For the plasma samples total RNA was extracted using Plasma/Serum RNA Purification Midi Kit (Norgen Biotek Corp.) and concentrated and purified further using RNA Clean-up and Micro-Elute Kit (Norgen Biotek Corp.), following the manufacturer’s instructions. From the four tissue samples, a small piece of approximately 5 mg was used where it was initially homogenized and then sonicated to further disrupt the cells of the tissue. Total RNA was isolated, from each sample, using miRCURY RNA Isolation Kit-Tissue (Exiqon), in accordance with the manufacturer’s instructions. The A260/280 ratio and the A260/230 ratios for the purified RNA was measured on a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific). These data are summarized in Supplementary Table S1. To monitor for hemolysis, an RNA sample extracted from a hemolyzed blood sample was used as a control. The level of the erythrocyte specific hsa-miR-451 was compared between this control sample with each of the extracted plasma samples prior to any experimental work undertaken. This hemolysis indicator allowed for the elimination of samples with high expression levels of hsa-miR-451 [26 (link)].

For miRNA microarray analysis, tissues were harvested on post-fracture day 14 and stored in RNAlater (Ambion, Austin, TX, USA). Total RNA, including miRNA, was extracted from the tissue specimens of five different animals in each group using a miRCURY RNA Isolation Kit-Tissue (Exiqon, Vedbaek, Denmark). The quality of the total RNA was verified using an Agilent 2100 Bioanalyzer profile (Agilent Technologies, Santa Clara, CA, USA). One microgram of total RNA from sample and reference RNAs was labeled with Hy3 and Hy5 fluorescent labels, respectively, using a miRCURY LNA microRNA Hi-Power Labeling Kit (Exiqon). The Hy3-labeled samples and a Hy5-labeled reference RNA sample were mixed pair-wise and hybridized to a miRCURY LNA Array, version 6^th Generation (Exiqon), which contains capture probes targeting all human, mouse, and rat miRNAs registered in miRBASE version 16.0. Labeling, hybridization, washing, and scanning were performed following the instructions of the manufacturers. Data analysis was carried out using Feature Extraction 10.7.3.1 (Agilent Technologies).

The total RNA for the specimens was isolated using miRCURY™ RNA Isolation Kit–Tissue (Exiqon, Vedbaek, Denmark), and the total RNA from cells was extracted using RNAiso Plus (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. MiRNAs were reverse transcribed to cDNA using All-in-One™ miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia, Guangzhou, China). The real-time quantitative PCR (qPCR) of miRNAs was performed with All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia). We synthesized mRNAs to cDNA using Bimake™ All-in-One cDNA Synthesis SuperMix (Bimake, Houston, USA), and the qPCR of mRNAs was performed with Bimake™ 2x SYBR Green qPCR master mix (Bimake). Relative expression was calculated with the 2^−ΔΔCT method and levels were normalized using U6 and GAPDH for miRNAs and mRNAs, respectively. The primer sequences used in this study are shown in Table 2.