Comment[ArrayExpressAccession] E-GEOD-44795 MAGE-TAB Version 1.1 Public Release Date 2013-03-02 Investigation Title Real-time quantitative PCR analysis of spleen cells Comment[Submitted Name] Real-time quantitative PCR analysis of spleen cells Experiment Description TGF-M-NM-21 signaling pathway of spleen of the Gata1low mouse model of myelofibrosis Four condition experiment. Biological replicates: 3 control CD1 mice, 3 Gata1low mice, 3 SB431542-treated Gata1low mice and 3 Vehicle-treated Gata1low mice. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name ciaffoni Zingariello Martelli Ciaffoni Masiello Ghinassi D'Amore Massa Sancillo Li Goldberg Rana Migliaccio Person First Name fiorella Maria Fabrizio Fiorella Francesca Barbara Emanuela Margherita Laura Xiaochun Judith Rosa Anna Person Mid Initials D A R Person Email fiorella.ciaffoni@iss.it Person Affiliation Istituto Superiore di SanitM-CM- Person Phone +390649903074 Person Address Cellular Biology and Neurosciences, Istituto Superiore di SanitM-CM- , Viale Regina Elena 299, Rome, Italy, Italy Person Roles submitter Protocol Name P-GSE44795-1 P-GSE44795-4 P-GSE44795-5 P-GSE44795-2 P-GSE44795-3 P-GSE44795-6 Protocol Description Three different mice were analyzed per experimenalt point (replicate 1, 2 and 3). Differences in gene expression among groups were identified using the RT 2 Profiler PCR Array Data Analyses Web portal (QIAGEN) and considered statistically significant with a p<0.05 by two-tailed, StudentM-bM-^@M-^Ys t-test. For the normalization it was used the average of Gapdh as housekeeping gene. Target gene signals (Ct value cut-off 35) normalized to housekeeping gene; 2^-(Ave deltaCt), where deltaCt = (Ct_Target M-bM-^HM-^R Ave(Ct_HKG). The web-based software package automatically performs all deltadeltaCt based fold-change calculations from the uploaded raw threshold cycle data. Biological consequences of gene expression alterations were predicted with the David Bioinformatic Database (David Bioinformatics Resources 6.7 NIAID/NIH) Matrix normalized worksheet reports normalized signal (against housekeeping genes). Fold Change worksheet reports test/control (i.e., Group1/Control Group) ratios. Biological consequences of gene expression alterations were predicted with the David Bioinformatic Database (David Bioinformatics Resources 6.7 NIAID/NIH) ID_REF = VALUE = 2^(-Avg.(Delta(Ct)) PCR assays were performed using the TGFM-NM-2/BMP Signaling Pathway PCR array (PAMM-035A - SABiosciences, Frederick, MD). Reverse transcription was performed from 100 ng total RNA using RT2 First Strand kit (SABiosciences, Frederick, MD) following manufacturer's instructions. RT-PCR was performed with RT2 SYBR ROX qPCR Mastermix(SABiosciences, Frederick,MD) following manufacturer's instructions. Absolute quantification was detected by 7300 Real-TimePCR System (Applied Biosystem).Termal cycling conditions were setted following manufacturer's instructions n/a Mice were injected intraperitoneally with TGF-M-NM-2 Signaling inhibitor SB431542 (60 M-5g/kg/day) or vehicle (same volume) for two cycles of five consecutive days two days apart, rested for one month, and then treated for two additional cycles. Total spleen cells were lysed in Trizol. Total RNA extracted using Trizol following manufacturer's instructions. n/a Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name STRAIN CELL TYPE Experimental Factor Type strain cell type PubMed ID 23462118 Comment[SecondaryAccession] GSE44795 Comment[GEOReleaseDate] 2013-03-02 Comment[ArrayExpressSubmissionDate] 2013-03-01 Comment[GEOLastUpdateDate] 2013-06-05 Comment[AEExperimentType] transcription profiling by RT-PCR Comment[AdditionalFile:Data1] GSE44795_Fold-change_spleen.txt Comment[AdditionalFile:Data2] GSE44795_Non-normalized_spleen.txt SDRF File E-GEOD-44795.sdrf.txt