Unknown,Transcriptomics,Genomics,Proteomics

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MicroRNA expression profile in human gliomas

ABSTRACT: To investigate the regulatory mechanisms governing the malignant signature of different gliomas we analyzed microRNA expression profiles in human tumor samples of world health organization (WHO) grade I (benign tumors), II (low grade tumors) and IV (high grade tumors) and from primary cultures obtained from tumor samples of grade II and IV. Patients This study included tumor samples histologically verified as astrocytic gliomas obtained from patients who had undergone craniotomy for microsurgical tumor removal. According to the revised WHO classification, tumors were diagnosed as: grade I or pilocytic astrocytomas; grade II or diffuse fibrillary astrocytomas; grade IV or glioblastoma multiforme. Primary cell cultures from grade II and grade IV gliomas were also obtained and miRNA expression in these cultures were analyzed RNA extraction Total RNA, including small RNA, was isolated from tissue samples using the mirVanaTM miRNA Isolation Kit (Ambion) following the standard protocol. The quantity and quality of the purified RNA was evaluated by spectrophotometric analysis and electrophoresis on denaturing gel of acrylamide. Multiplex Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) The miRNAs were first converted to cDNA using Multiplex RT for TaqMan Array Human MicroRNA Panel. The RT Master mix included 100 mM each of dNTPs , 50 U/ml MultiScrabe reverse transcriptase (Applied Biosystems), 20 U/M-BM-5l RNase inhibitor (Applied Biosystems) and 10X RT Buffer. The 10 M-BM-5l reactions, including 7 M-BM-5l of RT master mix, 2 M-BM-5l of purified microRNA and 1 M-BM-5l of Multiplex RT Human primer pool (Applied Biosystem), were incubated in ice for 5 min and then in a thermal cycler for 30 min at 16M-BM-0C, 30 min at 42M-BM-0C, 5 min at 85M-BM-0C, and then hold at 4M-BM-0C. miRNA levels were normalized to the expression of small nucleolar RNAs, RNU44, RNU48 and RNU6B. All reverse transcriptase reactions, including no-template controls and RT controls, were run in duplicate. Real-time PCR was performed using a standard TaqMan PCR kit procedure on an M-bM-^@M-^\Real Time Fast 7900 HTM-bM-^@M-^] PCR System (Applied Biosystems). The 100 M-BM-5l PCR included 50 M-BM-5l RT product (before diluited 1:60) and 50 M-BM-5l TaqMan Universal PCR Master Mix (2X) (Applied Biosystems). The total volume were loaded into Card TaqMan Low Density Array Human MicroRNA Panel (Applied Biosystem) including a total of 384 human microRNAs publicated on databases www.sanger.ac.uk. The reaction cards was runned at 50M-BM-0C for 2 min and 95M-BM-0C for 10 min, followed by 40 cycles of 97M-BM-0C for 30s and 59,7M-BM-0C for 1 min. All reactions were run in triplicate. Analysis of data was performed using the SDS 2.3 software using the 2-M-bM-^HM-^FM-bM-^HM-^FCt (relative quantitative) method . The M-bM-^HM-^FCt of every miRNA was determined in relation to the endogenous control RNA U6 that was invariably expressed in all samples. The M-bM-^HM-^FM-bM-^HM-^FCt value was determined in relation to the calibrator, namely the normal brain tissue. Resulting data were grouped according to the tumor grading e selectioned using a cut-off value of 3. Results were expressed as M-bM-^@M-^\fold changeM-bM-^@M-^] over normal brain tissue. We analyzed two samples of grade I, two samples of grade II, two samples of grade IV gliomas. Four samples form norma brain were used as norma control. Primary cell cultures form grade II and grade IV samples were used for the analysis. All reverse transcriptase reactions, including no-template controls and RT controls, were run in triplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Alfredo Conti

PROVIDER: E-GEOD-50924 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Preparation of nuclear extracts D. melanogaster Schneider cells were prepared as previously described by Andrews and Faller (1991). Briefly, in a typical preparation, 8x106 cells (grown in Schneider medium plus 10% fetal calf serum; GIBCO-BRL) were pelleted and resuspended in 1,5 mL cold PBS1X; the cell suspension is then tranferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 M-5L cold Buffer A (10mM HEPES-KOH pH 7.9 at 4M-0C, 1.5mM MgCl2, 10mM KCl, 0.5mM duthiothreitol, 0.2mM PMSF, 0.2U/microL RNasin). The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 100 M-5L of cold Buffer C (20mM HEPES-KOH pH 7.9, 25% glycerol, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM dithiothreitol, 0.2mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 min at 4M-0C and the supernatant fraction is stored at M-^V70M-0C. All buffers were prepared from double-distilled autoclaved water that had been treated with 0.1% DEPC (Sigma). RNA immunoprecipitation and Reverse Transcription For RNA immunopreciptation, the nuclear extract was incubated whit 50M-5g of monoclonal C1A9 anti HP1 antibody and the mixture was kept at 4M-0C overnight under continuous gentle movement. One-hundred microliters ofprotein G-Sepharose (Sigma) suspension (50% packed Sepharose in Buffer C) was added and the incubation was continued overnight as described.The beads were pelleted by 2 min centrifugation at 240 g at 4M-0C; the pellet was washed briefly three times whit each 1mL of IP Wash Solution (150mM NaCl, 50mM Tris pH 7.5, 0.5% NP40) and the Sepharose was transferred for eluition into a fresh plastic tube , pelleted again and then the supernatant was removed completely. To elute the immunocomplexes for protein analysis, an aliquot of beads were suspended in 50M-5L SDS-PAGE sample buffer and incubated for 10 min at 90M-0C; following centrifugation the supernatant was removed and used for Western blot for testing the presence of HP1 protein. To elute the immunoprecipitated RNAs, the pelleted beads were boiled in 200M-5L of DEPC wather for 5 min, spun, and the supernatant recovered; 1mL of Trizol (Invitrogen) was added to 200 M-5L of supernatant and mixed well, followed by the addition of 200M-5L of chloroform. This mixture was incubated at 4M-0C for 5 min and then centrifuged at 12000 g for 15 min; the RNAs in the aqueous phase was precipitated whit half volume of isopropanol; after precipitation, the RNAs was resuspend in 10M-5L of DEPC wather. Contaminating DNA was digested whit RNase-free DNase I (Sigma). The RNA purified from the previous step is used as a template to synthesize cDNA using oligo dT , random hexamers and SuperScript reverse transcriptase III (Invitrogen) according to the manufacturer's protocol. cDNA amplification and labeling The cDNA was used as template for a two-step random PCR amplification; in Round A, Sequenase is used to extend randomly annealed primers (Primer A) to generate templates for subsequent PCR; during Round B, the specific primer B is used to aplify the templates previously generated and finally round C consist of additional PCR cycles to inorporate the amino allyl dUTP nucleotide. About 25 ng of each cDNA sample was used for two 8 min extension with 2,7 mM Round A primer (5M-^R-GTT TCC CAG TCA CGA TCN NNN NNN NN-3M-^R, N being a mixture of all four nucleotides with 60% A+T and 40% G+C) at 37M-0C with 267U/ml Sequenase version 2.0 (usb). DNA was denatured at 94M-0C for 2 min and cooled to 10M-0C , and Sequenase 2.0 was added between extensions. The resulting products were used as template for 25 cycles of PCR using 1U/100M-5l Taq polymerase (Platinum Taq Invitrogen) and 10mM Round B primer (5M-^R-GTT TCC CAG TCA CGA TC-3M-^R). Finally this DNA was used as template for 25 cycles PCR to inorporate the amino allyl dUTP nucleotides to which the fluorescent dye may then be attached. To remove Tris buffer which interferes with the indirect coupling, the aminoallyl-cDNA samples were desalted by filtering through a Microcon M-^V30 and then mixed with the succinimidyl esters of the Cy3 or Cy5 dyes (Amersham Biosciences) in 0,1M sodium bicarbonate buffer (pH 9); the coupling reaction was icubated overnight in the dark at room temperature. Each dye labeled sample was purified by AutoSeq MicroSpin G-50 columns (Amersham Biosciences) following the manufacterers directions. Microarrays Hybridization and washing The dried labeled cDNAs pellet were resuspended in 80M-5l of DIG Easy Hyb (Roche) hybridization buffer containg 0,5 mg/ml yeast tRNa and 0,5 mg/ml salmon sperm DNA. Finally the DNA probe was denaturated by incubation at 65M-0C for 10 min and, after a brief centrifugation to spin down the drops, the mixture was pipetted onto a microarray; a coverslip was applied and the slide was placed in a microarray hybridation chamber (BioRad) and incubated overnight at 37M-0C. After hybridization, the slide was submerged in 0,01% SDS, 1%SSC until the coverslip slipped off the surface; The slide was transferred to a solution of 1%SSC and shaken at 50M-0C for 10 min, then washed once more by shaking in 0,1%SSC. Slide was dried by centrifugation for 3 min at 550 rpm and scanned with an ScanArray Lite Microarray Scanner (Packard Bioscience) with laser intensities chosen to maximize signals while avoiding pixel saturation. ScanArray express softwere was used to quantify hybridation signals; bad spots were flagged automatically by softwere and subsequently each slide was inspected manually.

Project description:To investigate the regulatory mechanisms governing the malignant signature of different gliomas we analyzed microRNA expression profiles in human tumor samples of world health organization (WHO) grade I (benign tumors), II (low grade tumors) and IV (high grade tumors) and from primary cultures obtained from tumor samples of grade II and IV. Patients This study included tumor samples histologically verified as astrocytic gliomas obtained from patients who had undergone craniotomy for microsurgical tumor removal. According to the revised WHO classification, tumors were diagnosed as: grade I or pilocytic astrocytomas; grade II or diffuse fibrillary astrocytomas; grade IV or glioblastoma multiforme. Primary cell cultures from grade II and grade IV gliomas were also obtained and miRNA expression in these cultures were analyzed RNA extraction Total RNA, including small RNA, was isolated from tissue samples using the mirVanaTM miRNA Isolation Kit (Ambion) following the standard protocol. The quantity and quality of the purified RNA was evaluated by spectrophotometric analysis and electrophoresis on denaturing gel of acrylamide. Multiplex Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) The miRNAs were first converted to cDNA using Multiplex RT for TaqMan Array Human MicroRNA Panel. The RT Master mix included 100 mM each of dNTPs , 50 U/ml MultiScrabe reverse transcriptase (Applied Biosystems), 20 U/µl RNase inhibitor (Applied Biosystems) and 10X RT Buffer. The 10 µl reactions, including 7 µl of RT master mix, 2 µl of purified microRNA and 1 µl of Multiplex RT Human primer pool (Applied Biosystem), were incubated in ice for 5 min and then in a thermal cycler for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then hold at 4°C. miRNA levels were normalized to the expression of small nucleolar RNAs, RNU44, RNU48 and RNU6B. All reverse transcriptase reactions, including no-template controls and RT controls, were run in duplicate. Real-time PCR was performed using a standard TaqMan PCR kit procedure on an “Real Time Fast 7900 HT” PCR System (Applied Biosystems). The 100 µl PCR included 50 µl RT product (before diluited 1:60) and 50 µl TaqMan Universal PCR Master Mix (2X) (Applied Biosystems). The total volume were loaded into Card TaqMan Low Density Array Human MicroRNA Panel (Applied Biosystem) including a total of 384 human microRNAs publicated on databases www.sanger.ac.uk. The reaction cards was runned at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 97°C for 30s and 59,7°C for 1 min. All reactions were run in triplicate. Analysis of data was performed using the SDS 2.3 software using the 2-∆∆Ct (relative quantitative) method . The ∆Ct of every miRNA was determined in relation to the endogenous control RNA U6 that was invariably expressed in all samples. The ∆∆Ct value was determined in relation to the calibrator, namely the normal brain tissue. Resulting data were grouped according to the tumor grading e selectioned using a cut-off value of 3. Results were expressed as “fold change” over normal brain tissue.

Project description:High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). Here we investigated how 4EGI-1, a small inhibitor of cap-dependent translation, induces apoptosis in T-ALL clones displaying hyperactive PI3K-AKT-mTOR signaling. 4EGI-1 treatment reduced the ribosomal occupancy of transcripts that define the molecular difference between T-ALL blasts and normal thymocytes. These transcripts encoded proteins for the translational machinery, for mitochondrial metabolism as well as oncoproteins such as Cyclin D1, Bcl-2 and c-myc. Therefore, targeting translation initiation proves valuable in situations where mTOR is activated by multiple events. For each biological replicate 5 x 107 #483 T-ALL cells were treated with 100 M-NM-<M 4EGI-1 or DMSO. After 2 h incubation at 37M-BM-0C cells were washed in ice-cold PBS containing cycloheximide (0.1 mg/ml), pelleted and resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 0.5 mM DTT, 5 mM MgCl2, 0.5 % nonidet-P40, 0.1 mg/ml cycloheximide, 10000 IU/ml dalteparin sodium) and incubated for 5 min on ice. All subsequent steps were carried out in the cold. After centrifugation for 10 min at 12000 x g, 0.4 ml supernatant was layered onto a 12 ml linear sucrose gradient (10 M-bM-^@M-^S 50% sucrose [w/v] in extraction buffer without nonidet-P40) and centrifuged at 4 M-BM-0C in an SW40Ti rotor (Beckman) at 35000 rpm without brake for 120 min. The gradients were harvested and absorbance profiles at 254 nm recorded (type 11 optical unit/UA-6 detector, ISCO). 1 ml fractions were collected and supplied with 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol for overnight precipitation at -20 M-BM-0C. RNA was purified using Nucleospin RNAII tubes (Macherey & Nagel) following the manufacturerM-BM-4s protocol. For microarray analysis RNA from polysome fractions were pooled, precipitated by adding LiCl to a final concentration of 2 M and resolubilized in 15 M-BM-5l of RNase-free water. RNA concentrations were determined and the samples were stored at -80 M-BM-0C. The cytosolic/total- RNA sample for normalization was obtained from the same samples (106 treated/non-treated cells) using the standard Trizol protocol.

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MicroRNA expression profile in human gliomas

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