Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Application of Serial Analysis of Gene Expression to the study of the gene expression profile of Leishmania infantum chagasi promastigote

ABSTRACT: 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).

ORGANISM(S): Leishmania chagasi

SUBMITTER: ADELINO LIMA NETO

PROVIDER: E-GEOD-29369 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:We determined the genome-wide digital gene expression (DGE) profiles of primary acute lymphoblastic leukemia (ALL) cells from 28 patients and fractionated blood cells from healthy blood donors taking advantage of “second generation” sequencing technology. The patients included in the study represent distinct subtypes of B-cell precursor (BCP) ALL and T-cell lineage ALL (T-ALL) and the controls are fractionated CD19+ and CD3+ cells. Gene expression analysis of 28 ALL patient samples with different immunophenotypic backgrounds including T-ALL (n=4) and patients with BCP ALL with diverse cytogenetic backgrounds: High Hyperploidy (HeH) (n=10), t(9;22) BCR-ABL1 (n=3), t(12;21) ETV6-RUNX1 (n=4), dic(9;20) (n=3), t(1;19)TCF3-PBX1, MLL/11q23 (n=1) and undefined/non-recurrent aberrations (n=1). Fractionated b-cells (CD19+) and t-cells (CD3+) isolated from peripheral blood of healthy donors were used as controls. Sequencing libraries were prepared from 1 µg of total RNA using reagents from the NlaIII Digital Gene Expression Tag Profiling kit (Illumina Inc, San Diego, CA, USA). mRNA was captured on magnetic oligo(dT) beads and reverse transcribed into double-stranded cDNA. The cDNA was cleaved using the restriction enzyme NlaIII. An adapter sequence containing the recognition sequence for the restriction enzyme MmeI was ligated to the NlaIII cleavage sites. The adapter-ligated cDNA was digested with MmeI to release the cDNA from the magnetic bead, while leaving 17 base-pairs of sequence in the fragment. The fragments were dephosphorylated and purified by phenol-chloroform. A second adapter was ligated at the MmeI cleavage sites. The adapter-ligated cDNA fragments were amplified by PCR, and the PCR products were purified on a 6% polyacrylamide gel. The ~96 base pair PCR products were excised from the gel and eluted overnight, followed by ethanol precipitation and re-suspension. Purified libraries were quality controlled and quantified on a Bioanalyzer using DNA 1000 series or High Sensitivity chips. The DGE libraries were diluted to a 10 nM concentration and sequenced on one lane of an Illumina GAII or GAIIx for 18 cycles.

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Application of Serial Analysis of Gene Expression to the study of the gene expression profile of Leishmania infantum chagasi promastigote

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