Comparison of microarray and RNA sequencing platforms for profiling microRNAs in formalin-fixed, paraffin-embedded human lung adenocarcinoma specimens
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ABSTRACT: MicroRNAs are useful biomarkers for various disease states, and their preservation in formalin-fixed, paraffin-embedded (FFPE) tissue makes them particularly useful for clinicogenetic studies. Although global microRNA expression in FFPE samples is routinely measured with microarrays, the utility of RNA sequencing for such profiling has yet to be established. In this study, to appraise the suitability of RNA sequencing, microRNAs in RNA from pathologic stage I lung adenocarcinoma FFPE samples were quantified with 8x60K Agilent® SurePrint™ G3 Human miRNA 8x60k (release 16.0) microarray and Illumina® HiSeq™ 2000 sequencing platforms.
Project description:We performed expression profiling using microarray technology. The expression profiling identified genes or mechanism potentially regulating the proliferation of human lung cancer cells by BoxA of HMGB1 and Alu-siRNA transfection. The RNAs of our experiment were hybridized with Agilent SurePrint G3 Human GE 8X60K, V3 Microarrays (Agilent®).
Project description:Sixth generation Exiqon® locked nucleic acid miRCURY™ LNA microarrays were used to profile in duplicate the expression of microRNAs in 250 or 400 ng of RNA isolated from a laser microdissectate of a formalin-fixed and paraffin-embedded human non-small cell lung cancer tumor. Laser microdissection was performed on a hematoxylin-eosin-stained, 8 um-thick section of formalin-fixed and paraffin-embedded tissue. The microdissectate was treated with proteinase K overnight at 55 ºC. Total RNA from the lysate was then extracted using FFPE RNA Isolation Kit from Norgen Biotek® (Thorold, Canada). RiboGreen assay was used to quantify RNA in the preparation. Two-hundred-fifty or 400 ng of RNA was used in duplicate for dual-channel microarray-based microRNA expression profiling. Sample RNA was labeled with the Cy3-like Hy3™ dye, mixed with a human universal reference RNA (product number AM6000, Ambion®, Austin, TX) labeled with the Cy5-like Hy5™ dye, and hybridized to the two-color miRCURY™ arrays.
Project description:Tissue samples were collected from 61 stage I/II PDAC cases then were placed into 10% neutral buffered formalin solution, fixed at roomtemperature (8–72 h) and processed routinely into a paraffin embedded tissue block (FFPE). The RNAstorm™ Kit (biotium, USA) was used to extracted and isolated total RNA fraction from FFPE Tissue Samples. RNA libraries were prepared for sequencing using prepared SMARTer® Stranded Total RNA-Seq Kit - Pico Input Mammalian Kit. RNA-seq was performed by the Illumina sequencing platform on a 150-bp paired-end run.
Project description:To evaluate genetic profile in neuroblastoma stage 3 fresh frozen or paraffin embedded (FFPE) tumor samples were dedicated to array comparative genomic hybridization (aCGH) for analyzing copy number variations. It was used 200 ng of tumor DNA for aCGH analyses. The Agilent SurePrint G3 CGH ISCA v2 Microarray Kit 8x60K array platform was used for genome evaluation without enzymatic digestion. The resolution for aCGH evaluation was established on 0.15Mb for frozen samples and 1Mb for FFPE.
Project description:'miRNA expression in 39 human AML samples was analyzed on Sureprint G3 Human miRNA (release 16.0) 8x60K Microarrays from Agilent Technologies. Half the samples represent primary AMLs while the other half represents secondary AMLs. Sample material consisted of cryopreserved bone marrow or blood aspirates. RNA from the samples was purified using the miRNeasy Mini Kit from Qiagen and the sample quality was tested on a Bioanalyser (all sample had RIN values ≥6.0 [mean=8.4]). The samples were labelled with Cy3 and processed according to Agilent''s protocol using vacuum concentration to dry the samples before microarray hybridization. The processed microarrays were scanned at 532 nm using a GenePix 4400A laserscanner at a resolution of 2.5 µm and the GenePix Pro 7.0 software.'
Project description:Biological effects of overexpression of miR-146b microRNAs in the A549 human lung cancer cell-line was studied. A549 cells were engineered to express the precursor RNA (pre-miR-146b) that generates the miR-146b microRNAs. Control cells were engineered using the same gene expression plasmid (pLemiR, Open Biosystems®) but without the pre-miR-146b insert. The Trans-Lentiviral GIPZ™ packaging system (Open Biosystems®) was used to generate stable transfectant populations of the engineered cells.
Project description:Fifth generation Exiqon® locked nucleic acid miRCURY™ LNA microarrays were used to profile the expression of microRNAs in whole blood of patients with non-small cell lung adenocarcinoma and of clinically relevant controls without the disease.