Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of the mouse transcriptome by Serial Analysis of Gene Expression

ABSTRACT: This study characterizes the expressed transcripts of 15 intact tissues in mice by using the serial analysis of gene expression (SAGE) strategy which indicates the relative level of expression for each transcript matched to the tag. Keywords: double-strand cDNA , serial analysis of gene expression (SAGE) , ditags Total RNA was isolated from tissues by using the RNA extraction kit (TRIzol Reagent, Invitrogen Canada Inc., Burlington, ON). Approximately 5 µg of mRNA was extracted with Oligotex mRNA Mini Kit (Qiagen Inc., Mississauga, ON). The SAGE method was performed as previously described (Velculescu et al., 1995; St-Amand et al., 2001). In brief, double-strand cDNA was synthesized from the mRNA using a biotinylated (T)18 primer and cDNA synthesis kit (Invitrogen Canada Inc.). The cDNA libraries were digested with the restriction enzyme NlaIII (New England Biolabs Inc., Pickering, ON). The 3'-terminal cDNA fragments were captured using streptavidin-coated magnetic beads (Dynal, Biotech LLC, Brown Deer, WI). After ligation of 2 annealed linker pairs, the cDNA fragments were digested with BsmFI (New England Biolabs Inc.). The blunting kit from Takara Bio Inc. (Otsu, Japan) was used for the blunting and ligation of the two tag populations. The resulting ligation products were amplified by PCR and digested with NlaIII. The band containing the ditags was extracted from the 12% polyacrylamid gel. Using T4 ligase (Invitrogen Canada Inc.), the ditags were self-ligated to form concatemers that were cloned into SphI site of pUC19. White colonies were screened by PCR and agarose gel to select long inserts for automated sequencing (Applied Biosystems 3730, Foster City, CA). The sequence and occurrence of each tag were analyzed by the SAGEana program, which is a new version of SAGEparser.pl (Dinel et al., 2005).

ORGANISM(S): Mus musculus

SUBMITTER: Kouamé Ettienne Kouadjo

PROVIDER: E-GEOD-5915 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Housekeeping and tissue-specific genes in mouse tissues.

Kouadjo Kouame E KE Nishida Yuichiro Y Cadrin-Girard Jean F JF Yoshioka Mayumi M St-Amand Jonny J

BMC genomics 20070522

<h4>Background</h4>This study aims to characterize the housekeeping and tissue-specific genes in 15 mouse tissues by using the serial analysis of gene expression (SAGE) strategy which indicates the relative level of expression for each transcript matched to the tag.<h4>Results</h4>Here, we identified constantly expressed housekeeping genes, such as eukaryotic translation elongation factor 2, which is expressed in all tissues without significant difference in expression levels. Moreover, most of ...[more]

PMID: 17519037

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Project description:MM-CM-<ller cells are the principal glial cells in the retina. Alterations in MM-CM-<ller cell behaviour are observed in retinal tissue from patients with proliferative diabetic retinopathy. The purpose of this study was to compare gene and protein expression profile of normal human MM-CM-<ller cells (NHMC) with two spontaneously human MM-CM-<ller cell lines generated from type 1 (HMCL-I) and type 2 (HMCL-II) diabetic donors using Serial Analysis Gene Expression (SAGE). Approximatively 50 000 tags were sequenced for each of the three SAGE libraries. Identification of the transcripts was obtained by matching the 15bp (CATG + 11bp) with the UniGene and GenBank databases. Classification of the genes was based upon the updated information of the genome directory found at the NCBI website. SAGE allowed us to characterize the entire transcriptome of human MM-CM-<ller cells and to compare these data with those from MM-CM-<ller cells of type 1 and 2 diabetic donors. Polyadenylated RNA was extracted with the Oligotex mRNA Purification System (Qiagen), annealed with the biotin-5 -T18-3 primer and converted to cDNA with the cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The resulting cDNA library was digested with NlaIII (anchoring enzyme), and the 3M-bM-^@M-^Y restriction fragments were isolated with streptavidin-coated magnetic beads (Dynal Biotech, Oslo, Norway) and separated into two populations. Each population was ligated to one of the two annealed linker pairs and extensively washed to remove unligated linkers. The tag beside the most 3M-bM-^@M-^Y NlaIII restriction site (CATG) of each transcript was released by digestion with BsmFI (tagging enzyme). The blunting kit from Takara Co. (Otsu, Japan) was used for the blunting and ligation of the two tag populations. The resulting ligation products containing the ditags were amplified by PCR with an initial denaturation step of 1 min at 950C, followed by 22 cycles of 20 s at 940C, 20 s at 600C and 2 s at 720C with 27 bp primers. The PCR product was digested with NlaIII, and the band containing the ditags was extracted from the acrylamide gel. The purified ditags were self-ligated to form concatemers. The concatemers of 500 to 1800bp were isolated by agarose gel. The resulting DNA fragments were ligated into the SphI site of pUC19 and cloned into UltraMAX DH5M-oM-^AM-!FT (Invitrogen). White colonies were screened by PCR to select long inserts for automated sequencing. Sequence files were analyzed by the SAGEana program, a modification of SAGEparser (ftp://ftp.pbrc.edu/public/eesnyder/SAGE/). Tags corresponding to linker sequences were discarded, and duplicate concatemers were counted only once. We used the comparative count display (CCD) test to identify the transcripts that were differentially expressed significantly (P<0.05) between the groups with more than a twofold change. The CCD test makes a key-by-key comparison of two key-count distributions by generating a probability that the frequency of any key in the distribution differs by more than a given fold factor from the other distribution.

Project description:This study describes the application of the LongSAGE methodology to study the gene expression profile in Leishmania infantum chagasi. A tag library was created using the LongSAGE method and consisted of 14,208 tags of 17 bases. Of these, 8,427 (59.3%) were distinct. BLAST research of the 1,645 most abundant tags showed that 12.8% of them identified the coding sequences of genes, while 82% (1,349/1,645) identified one or more genomic sequences that did not correspond with open reading frames. Only 5.2% (84/1,645) of the tags were not aligned to any position in the L. infantum genome. The UTR size of Leishmania and the lack of CATG sites in some transcripts were decisive for the generation of tags in these regions. LongSAGE revealed the gene expression profile in promastigotes of L. i. chagasi. Additional analysis will allow a better understanding of the expression profile and discovering the key genes in this life cycle. The I-SAGETM Long kit (Invitrogen) was used to construct the L. i. chagasi library according to the manufacturer's recommendations. The following were included among the main steps: polyadenylates mRNAs were captured by oligo (dT) linked to magnetic beads and used for cDNA synthesis. The cDNA was digested with 60 U of NlaIII (anchoring enzyme) and the 3' ends of the cDNAs were isolated using the beads. The resulting cDNA 3' was divided into two equal portions and connected to two LongSAGE adapters, A and B. The long tags were released by the enzyme MmeI and linked to form ~130 bp ditags. Dilutions of 1:40 of the product were amplified in 27 PCR cycles (300 reactions in total). The precipitated PCR products were separated on 12% polyacrylamide gel and the 130 bp bands were cut out and the precipitated DNA was digested with NlaIII. The digestion products were separated on 12% polyacrylamide gel and ~34 bp ditags were cut out and linked to form concatemers. The concatemers were separated on 6% polyacrylamide gel and the fractions of 250-500 and 500-800pb were isolated and cloned into pZErO-1 vector digested with SphI. Vectors with concatemers were cloned in E. coli DH10b by electroporation and the isolated recombinant vectors were used as template for sequencing in the ABI Prism 3100 (Applied Biosystems).

Project description:Ovarian folliculogenesis in mammals is a complex process involving cross talk between germ and somatic cells. Carefully orchestrated expression of transcription factors, cell adhesion molecules and growth factors are required for success. We have identified a germ-cell specific, basic helix-loop-helix transcription factor, FIGLA (Factor In the GermLine, Alpha) and demonstrated its involvement in two independent developmental processes: formation of the primordial follicle and coordinate expression of zona pellucida genes. Taking advantage of Figla null mouse lines, we have used a combined approach of microarray and Serial Analysis of Gene Expression (SAGE) to identify potential downstream targets genes. Using high stringent cutoffs, we find that FIGLA functions as a key regulatory molecule in coordinating expression of the NALP family of genes, genes of known oocyte-specific expression and a set of functionally un-annotated genes. Keywords: Serial Analysis of Gene Expression The libraries were constructed using Sau3AI as the anchoring enzyme as described in the SAGE protocol (Cheval et al., 2002). Total RNA (~50 µg) was obtained from newborn normal and Figla null ovaries with an RNeasy mini kit (Qiagen, Valencia, CA, USA). The presence of MSY2 and the absence of ZP2 transcripts as detected by one-step qRT-PCR (Qiagen, Valencia, CA, USA) confirmed the identity of Figla null ovaries. Poly(A)+ RNA was isolated with oligo d(T)-conjugated magnetic beads (Dynal-Invitrogen, Calsbad, CA, USA). SAGE libraries are constructed as per the protocol described by Virlon B and group (Virlon B et al, 1999).Briefly, cDNA was synthesized from 5 μg of poly(A) RNAs with the cDNA synthesis system kit. Double stranded cDNA was synthesized using the Invitrogen SuperscriptII kit. The double-strand cDNA was digested with Sau3AI, and the 3′ end was isolated through binding to 1 mg of paramagnetic streptavidin beads (Dynal, Invitrogen, Calsbad, CA, USA), The streptavidin-bound cDNA was divided into two fractions. Each fraction was ligated to 10 pmol of either linker 1 or linker 2, then digested with BsmFI. The released cDNA tags were blunt-ended for 10 min at 42°C in a 50-μl reaction volume containing 20 mM Tris HCl (pH 7.5), 10 mM MgCl2, 25 mM NaCl, 10 mM DTT, 400 μM dNTP, and 10 units of T7 DNA polymerase (Pharmacia Biotech). The two fractions then were ligated to each other by using T4 DNA ligase. PCR (95°C, 30 sec; 58°C, 30 sec; 70°C, 45 sec: 26–28 cycles) was carried out on 1% of ligation reaction in a 100-μl reaction volume containing 20 mM Tris HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 4 mM DTT, 100 μM dNTP, 50 pmol of primers 1 and 2, and 5 units of Taq polymerase. The products from 10–12 reactions were pooled. The 110-bp DNA fragment was purified by agarose gel electrophoresis and submitted to preparative PCR (120–150 reactions performed as described above, except that the amount of primers was reduced to 25 pmol and the number of cycles was 12). The PCR sample was digested with Sau3AI, and ditags were purified and concatenated as described (Velculescu et al, 1995). Concatenated ditags were cloned into Bluescript pKS (Stratagene, La Jolla, CA, USA) using the BamHI site and blue-white screening, prior to sequence, identified colonies with inserts. Plating, picking, DNA preparation and sequencing were performed at the the NIH Intramural Sequencing Center (NISC). Low quality sequence data were eliminated by Phred analysis.

Dataset Information

Characterization of the mouse transcriptome by Serial Analysis of Gene Expression

Publications

Housekeeping and tissue-specific genes in mouse tissues.

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