Specific taqman mirna assays

All animal experiments were performed in accordance with the research protocol approved by the Institutional Animal Care and Use Committee. The University has a file with the Office of Laboratory Animal Welfare and an approved Assurance Statement (#A3314-01). C3H/HeN mice (Charles River) were infected with 2.25 × 10⁵ pfu of R. conorii per animal administered intravenously (IV). The control animals received an IV injection of the identical volume of saline. Four animals were used per group in two independent experiments, i.e., n = 8. On day 3 post-infection, mice were anesthetized by inhalational isofluorane for collection of blood by cardiac puncture, after which the mice were euthanized and the lungs were removed aseptically and preserved in an RNAlater solution for isolation of total RNA and analysis by q-RT-PCR using miRNA-specific Taqman assays (Applied Biosystems, Waltham, MA, USA).

miRNA and mRNA were extracted from FFPE samples of colon tumor and normal tissue of patient carrying variant in 3′UTR of MSH2 gene using Qiagen miReasy FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA quantity was assessed using the Qubit RNA BR Assay Kit (Invitrogen, by Thermo Fisher Scientific, Eugene, Oregon, USA). The expression of miR-137 and MSH2 as well as the relative endogenous controls, U6 snRNA and GAPDH, respectively, were determined using TaqMan MicroRNA Reverse Transcription Kit and specific TaqMan miRNA assays (Taq-Man, Applied Biosystems, Inc.,Waltham, Massachusetts, Stati Uniti). All qRT-PCR assays for normal and tumor sample were performed in triplicate using the same amount of isolated RNA.

Quantification of mRNA expression was performed in triplicate using quantitative reverse transcription polymerase chain reaction (PCR) with the following primers: Forward, 5′-CTCCCATCTCAACTCCCTGA-3′ and reverse, 5′ACCAGCTTCCTGAACAGCTC-3′, for P2X7. Specific TaqMan^® miRNA Assays (Applied Biosystems Life Technologies, Foster City, CA, USA) were used for Let-7 g, miR-21, miR-205 and RNU6B according to the manufacturer’s instructions. The threshold cycle (Ct) and baselines were determined using manual settings, where Ct was set in the linear phase of the amplification and baseline in the initial cycles of PCR. Expression was calculated by relative quantification and fold expression changes were determined by the 2^−ΔΔCT method using the DataAssist software (Applied Biosystems Life Technologies).
Mutational analysis was also performed for all samples. The mutational status of the codons 12–13 of the K-Ras gene was analyzed by pyrosequencing using the anti-epidermal growth factor receptor (EGFR) MoAb response^® kit (K-Ras status) (Diatech Pharmacogenetics, Jesi, Italy) according to the manufacturer’s instructions.
PCR-single stranded conformation polymorphism and sequencing analysis were used for EGFR genotyping in exons 18–21, as previously described (14 (link)).

Hematoxylin and eosin (H&E) stained slides from FFPE samples were reviewed by the pathologist, who confirmed the diagnosis and selected areas representative of both HCTC and normal tissue. Two to three cores (1 mm in diameter) of histologically confirmed HCTC and of normal tissue were obtained from each specimen. For all the patients, miRNA was extracted from FFPE samples of primary tumor and of normal thyroid tissue using Qiagen miRNeasy FFPE Kit (Quiagen, Hilden, Germany) according to the manufacturer's instructions. Quality of RNA and its size were assessed using the Agilent Small RNA kit (Agilent Technologies, California, USA). The expression of six miRNAs (miR-138, miR-183, miR-221, miR-222, miR-768-3p, and miR-885-5p) and U6 snRNA as endogenous control was determined using TaqMan MicroRNA Reverse Transcription Kit and specific TaqMan miRNA assays (TaqMan, Applied Biosystems, California, USA). Reactions for all miRNA assays and for all normal and tumor samples were performed in triplicate using the same amount of isolated RNA. Specific miRNAs, which were analyzed in our study, were selected based on data from the literature [17 (link), 26 (link)–28 (link)].