Project description:Differences in the expression profile of hepatic and pancreatic stellate cells are investigated. Aim is to identify organ and disease specific transcriptome signatures of stellate cells, comparing hepatic and pancreatic stellate cells obtained from tissues of chronic inflammation, and primary or metastatic cancers of the pancreas. Tissues of chronic pancreatitis (n=6), pancreatic ductal adenocarcinoma (n=5), liver cirrhosis (n=5) and liver metastasis of pancreatic ductal adenocarcinoma (n=6) were collected and stellate cells were isolated by the outgrowth method. Using cDNA microarrays, differentially expressed genes are identified.
Project description:We used the TriFecta TM (IDT, Integrated DNA Technologies, USA) anti-Aire siRNA sequence (GGAUUCUCUUUAAGGACUACAAUCTAGAUUGUAGUCCUUAAAGAGAAUCCUC) to silencing Aire mRNA. Confluent cultures of 3.10 mTEC cell line were transfected with 10 nM of anti-Aire siRNA using Hiperfect reagent (Qiagen) following manufacturers instructions. <br><br>After transfection, cells were cultured during 24 h in RPMI medium as above mentioned and total RNA was extracted using the mirVana kit (Ambion), which served as template for cDNA synthesis. Gene knockdown was confirmed by semi-quantitative reverse transcription PCR (RT-PCR) using the primers 5prim CATCCTGGATTTCTGGAGGA 3prim forward and 5prim GCTCTTTGAGGCCAGAGTTG 3prim reverse, which allowed amplification of a 253 bp PCR product corresponding to a segment of the Aire mRNA (cDNA). The PCR mix contained 200 uM dNTPs, 1.5 mM MgCl2, 50 mM KCl, 10 mM TRIS-HCl (pH 8.4), 10 uM each primer and 2 U Taq DNA polymerase. The cycling temperature was: 35 x 30 sec each (94oC, 57oC and 72oC). <br><br>An anti-HPRT siRNA included in this kit but whose sequence was not furnished was used in parallel to evaluate the efficiency of the gene knockdown assay on 3.10 mTEC cell line (data not shown).<br><br>
Project description:Direct comparison of gene expression in 13.5dpc embryonic gonad/mesonephros between male Mrotm1H/Mrotm1H mice and male wild-type littermates. Two independent pools of 10 gonads (5 mice) were compared on 4 microarrays (two colourswaps).
Project description:Exploration of transcriptome expression in 5 control and 4 familial dysautonomia (FD) human olfactory ecto-mesenchymal stem cells (hOE-MSCs) at very early (P1 and P2) and later (P5 and P9) cell passages.
Project description:The 13,824 cDNA microarray was constructed by printing 6912 PCR-amplified cDNA clones in duplicate. These were derived from a previous EST study (Clark et al, 2007) and comprised 3840 clones from the Library D2 (fully dehydrated animals, cooled to -14°C) and 3072 clones from the Library D1 (dehydrated animals cooled to -2°C). The Stratagene SpotReport Alien Array Validation System (Stratagene, La Jolla, CA, USA) was included on the microarray. Construction and hybridization of the arrays were performed as described in Purac et al (2008) with the exception of the amino-modified primers used for the initial cDNA amplification for array printing, which in this study were: (pAL32FOR: TTCTCGGGAAGCGCG and M13 forward: GTAAAACGACGGCCAG). Hybridizations were performed using control animals in combination with the groups listed below.<br><br><br><br>Six groups of animals were used for the hybridizations. Treatments were as follows:<br><br>C = control = live animals from +5°C<br><br>M2 = cold dehydrated animals, cooled to -2°C at a rate of 2°C/week<br><br>M7 = cold dehydrated animals, cooled to -7°C at a rate of 2°C/week<br><br>H18 = animals taken from -7°C, and left to recover for 18 hours at 4°C with moisture.<br><br>Salt 0.9 = animals slowly dehydrated over a saturated solution of sodium nitrate (which gives a constant humidity of 98% RH) to produce animals with a similar water content as the M2 animals<br><br>Salt 0.2 = as for Salt 0.9, but the animals were dehydrated to a similar water content to the M7 animals.<br><br>6 biological replicates were performed for each condition, as well as some dye swaps.