Dataset Information

RNAseq of NTCU-induced mouse lung squamous cell carcinoma

ABSTRACT: Topical treatment with N-nitrosotris-(2-chloroethyl)urea (NTCU) induces lung squamous cell carcinoma in mouse. The aim of this study is to analyze the expression profile of the mouse NTCU-induced SCC compared to normal lung to identify gene expression changes in the SCC cells. NIH Swiss mice presenting lung SCC and control healthy mice were used for laser capture microdissection (LCM). LCM was performed in a LMD7000 microscope (Leica Microsystems GmbH, Wetzlar, Germany). RNA was extracted using the RNeasy FFPE kit (Qiagen, Hilden, Germany) and libraries were prepared from using the SMARTer Stranded Total RNA-Seq Kit – Pico Input Mammalian (Clontech Laboratories, Inc., Mountain View, CA, USA). Sequencing was performed using an Illumina HiSeq 2500 generating 50 base pair paired end reads. Reads of low quality were filtered, and adapters trimmed using Cutadapt v1.9.1. Alignment to the reference genome (UCSC mm10) was performed using TopHat v2.0.1, and RNA counts were generated for each gene using the Python package HTSeq v0.6.1, employing the “intersection-strict” overlap mode. DESeq2 was used to normalize and transform the data, examine the relationship between samples (by plotting the first two principal components) and to test for differential expression

ORGANISM(S): Mus musculus

PROVIDER: GSE129482 | GEO | 2019/12/31

REPOSITORIES: GEO

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Project description:The mammalian lens consists of an aged core of quiescent cells enveloped by a layers of mature fully elongated and younger, continuously elongating and transcriptionaly active cells. In the present study, we compared global gene expression profiles in elongating and mature, non-elongating fiber cells. The microarray-based comparison of gene expression profiles was performed using Agilent 22K Mouse Oligo microarrays with two rounds of RNA amplification from samples obtained by laser capture microdissection (LCM) of paraffin-embedded lens sections. Lenses were removed and immediately fixed using 4% paraformaldehide or methanol-based UMFIX reagent (Sakura Finetek USA, Inc.). To map the exact location of both elongating and maturing fibers on the lens slices, lenses were vibratome-sliced (Vibratome 1000, St. Louis, MO) as described previously and the GFP expression pattern was captured by confocal microscopy. For RNA extraction lens tissue was sliced into 5 micron-thick paraffin sections and microdissected using LCM (Leica Microsystems, Bannockburn, IL). The mid-saggital slices were used both for syncytium border measurements and for LCM. Control measurement (data not shown) confirmed that similar rates of shrinkage in paraformaldehyde- and UMFIX-fixed samples did not affect precision of LCM dissection. Fiber cell samples were dissected out of 5 micron-thick slices. In one experiment we typically processed about 40 slices, which was sufficient to collect the minimum of 200 zone-specific cells pooled from P5 littermate lenses. This sample size provided a reliable representation of RNA species in experimental procedure originally designed and tested for just 1-10 cells. Localization of the syncytium border characterized by the abrupt change of GFP labeling pattern was captured by confocal microscopy as described previously. In brief, GFP fluorescence was visualized using LSM510 instrument (Carl Zeiss, NY) equipped with an argon/krypton laser at 488 nm excitation and a 515-565 nm band pass emission filter. Physical parameters of the zones containing young and maturing fibers were measured in fixed lens slices using the morhphometric software provided by Zeiss. Lenses were fixed in 4% paraformaldehyde/PBS and sectioned with a vibratome. Cells from elongating and maturing fiber regions were dissected out using the Leica DMLA laser capture microscope, (Leica Microsystems, Bannockburn, IL). The cut-out pieces containing captured cells were put directly into tubes containing the lysis buffer supplied in the Absolutely RNA® nanoprep kit (Stratagene, La Jolla, CA). Total RNA from the microdissected tissue sections was extracted and purified using the Absolutely RNA nanoprep kit according to the manufacturer's protocol. Caps briefly placed onto the section without laser activation were used as negative controls. Samples from several age-matched lenses were pooled together to obtain differentiation-specific samples for microarray analysis. Target RNA amplification and labeling with Cy-3 or Cy-5 dyes from CyDye Post Labelling Reactive Dye Pack (Amersham, USA) was carried out in two rounds using the Amino Allyl MessageAmp™ aRNA Kit (Ambion, USA) as specified by the manufacturer. Quality and size distribution of the targets were determined by the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The Amino Allyl MessageAmp aRNA Kit is configured to incorporate the modified nucleotide, 5-(3-aminoallyl)-UTP (aaUTP) into the aRNA during in vitro transcription. Once purified and fragmented, the dye labeled aRNA was used for microarray hybridization. Labeled amplified RNA was hybridized to the 22K Mouse Oligo microarrays (Agilent Technologies)) according to the manufacturer's instructions. After hybridization microarrays were washed and scanned using GenePix 4000A (Axon Instruments,Inc.). Keywords = Lens Keywords = differentiation Keywords = microarray Keywords = gene expression profiling Keywords = laser capture microdissection Keywords = RNA amplification Keywords: repeat sample

Project description:The mammalian lens consists of an aged core of quiescent cells enveloped by a layers of mature fully elongated and younger, continuously elongating and transcriptionaly active cells. In the present study, we compared global gene expression profiles in elongating and mature, non-elongating fiber cells. The microarray-based comparison of gene expression profiles was performed using Agilent 22K Mouse Oligo microarrays with two rounds of RNA amplification from samples obtained by laser capture microdissection (LCM) of paraffin-embedded lens sections. Lenses were removed and immediately fixed using 4% paraformaldehide or methanol-based UMFIX reagent (Sakura Finetek USA, Inc.). To map the exact location of both elongating and maturing fibers on the lens slices, lenses were vibratome-sliced (Vibratome 1000, St. Louis, MO) as described previously and the GFP expression pattern was captured by confocal microscopy. For RNA extraction lens tissue was sliced into 5 micron-thick paraffin sections and microdissected using LCM (Leica Microsystems, Bannockburn, IL). The mid-saggital slices were used both for syncytium border measurements and for LCM. Control measurement (data not shown) confirmed that similar rates of shrinkage in paraformaldehyde- and UMFIX-fixed samples did not affect precision of LCM dissection. Fiber cell samples were dissected out of 5 micron-thick slices. In one experiment we typically processed about 40 slices, which was sufficient to collect the minimum of 200 zone-specific cells pooled from P5 littermate lenses. This sample size provided a reliable representation of RNA species in experimental procedure originally designed and tested for just 1-10 cells. Localization of the syncytium border characterized by the abrupt change of GFP labeling pattern was captured by confocal microscopy as described previously. In brief, GFP fluorescence was visualized using LSM510 instrument (Carl Zeiss, NY) equipped with an argon/krypton laser at 488 nm excitation and a 515-565 nm band pass emission filter. Physical parameters of the zones containing young and maturing fibers were measured in fixed lens slices using the morhphometric software provided by Zeiss. Lenses were fixed in 4% paraformaldehyde/PBS and sectioned with a vibratome. Cells from elongating and maturing fiber regions were dissected out using the Leica DMLA laser capture microscope, (Leica Microsystems, Bannockburn, IL). The cut-out pieces containing captured cells were put directly into tubes containing the lysis buffer supplied in the Absolutely RNAÂ® nanoprep kit (Stratagene, La Jolla, CA). Total RNA from the microdissected tissue sections was extracted and purified using the Absolutely RNA nanoprep kit according to the manufacturer's protocol. Caps briefly placed onto the section without laser activation were used as negative controls. Samples from several age-matched lenses were pooled together to obtain differentiation-specific samples for microarray analysis. Target RNA amplification and labeling with Cy-3 or Cy-5 dyes from CyDye Post Labelling Reactive Dye Pack (Amersham, USA) was carried out in two rounds using the Amino Allyl MessageAmpâ?¢ aRNA Kit (Ambion, USA) as specified by the manufacturer. Quality and size distribution of the targets were determined by the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The Amino Allyl MessageAmp aRNA Kit is configured to incorporate the modified nucleotide, 5-(3-aminoallyl)-UTP (aaUTP) into the aRNA during in vitro transcription. Once purified and fragmented, the dye labeled aRNA was used for microarray hybridization. Labeled amplified RNA was hybridized to the 22K Mouse Oligo microarrays (Agilent Technologies)) according to the manufacturer's instructions. After hybridization microarrays were washed and scanned using GenePix 4000A (Axon Instruments,Inc.).

Project description:MicroRNAs (miRNAs) are a novel class of short RNAs which have shown to be dysregulated in a variety of cancers including squamous cell carcinoma (SCC) of the head&neck. Microarray based miRNA expression profiles of cutaneous SCC (cSCC) however have not been investigated so far. Seven patients with cutaneous SCC were enrolled in the study. Tumor biopsies (n=7) were taken from the center of the tumor. Adjacent healthy skin (n=7) was biopsied as a control (intraindividual control). miRNA expression profiles of all specimen were detected by mircroarray miRNA expression profiling based on miRBAse 16 and compared. In 7 patients biopsy specimens of cutaneous SCC and control specimens from an adjacent healthy skin site near the tumor border (intraindividual control) were obtained during surgical removal of the tumor. Specimes were immediately stored in RNAlater (Qiagen, HIlden, Germany) at -80 M-BM-0C. Total RNA including miRNAs were isolated with miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturerM-bM-^@M-^Ys protocol. For RNA quality control purposes we determined RNA concentration, purity and RNA integrity number (RIN) with Agilent 2100 Bioanalyzer, RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA) and the NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). All of the following steps described were carried out according to the manufacturerM-BM-4s protocol. In order to enable assessment of labeling and hybridization efficiencies total RNA samples were spiked with MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA). After treatment with calf intestine phosphatase (CIP), a labeling reaction was started with 100 ng total-RNA per sample. For labeling dephosphorylated RNA, T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (miRNA Complete Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used. The Cyanine-3-labeled miRNA samples were then prepared for One-Color based hybridization (Complete miRNA Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA). Hybridization was performed at 55M-BM-0C for 20 hrs with Human miRNA Microarrays Release 16.0, 8x60K format (Agilent Technologies, Santa Clara, USA). Microarray slides were washed (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara) and dried with acetonitrile (Sigma-Aldich, St.Louis, USA). Fluorescent signal intensities were detected with Agilent DNA Microarray Scanner (Agilent Technologies, Santa Clara, USA) and Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA). Extraction of data was done by using the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA).

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RNAseq of NTCU-induced mouse lung squamous cell carcinoma

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