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Series GSE87128 Query DataSets for GSE87128
Status Public on Sep 21, 2016
Title Real-time quantitative PCR analysis of human acute myeloid leukemia primary cells
Organism Homo sapiens
Experiment type Expression profiling by RT-PCR
Summary Acute myeloid leukemia (AML) is a heterogeneous disease characterized by different recurrent cytogenetic and molecular aberrations that occur in hematopoietic progenitor cells and alter the growth, differentiation, and proliferation capacities of the progenitor cells. The treatment of AML has remained a huge challenge for oncohematologists. The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage is currently used in the treatment of acute myeloid leukemia. Here, we reasoned that DNA replication represents a less explored source of prognostic markers that could be used to predict AML prognosis and eventually provide potential targets for therapeutic targeting. Accurate execution of the DNA replication program limits cancer risk by preserving genome integrity. Multiple studies of solid cancers have provided evidence that defective or dysregulated DNA replication program triggers replicative stress, leading to the accumulation of genetic alterations. However, defective DNA replication as a source of markers in hematological malignancies has not been explored. Thus we explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy.
 
Overall design Patients samples Between Jan 1, 2000, and Dec 31, 2010, the use of fresh and thawed samples from 198 AML patients samples have been performed after informed consent and stored at the HIMIP collection (BB-0033-00060) of the Cancer Research Center of Toulouse. According to the French law, HIMIP collections have been declared to the Ministry of Higher Education and Research (DC 2008-307 collection 1) and we obtained a transfer agreement (AC 2008-129) after approbation by the “Comité de Protection des Personnes Sud-Ouest et Outremer II” (ethical committee). Clinical and biological annotations of the samples have been declared to the CNIL (Comité National Informatique et Libertés ie Data processing and Liberties National Committee), and these are supported by CAPTOR (Cancer Pharmacology of Toulouse-Oncopole and Region). This study was approved by the institutional review board (Ethical Committee of Research). Total RNA extraction and Reverse Transcription-PCR Total RNA were extracted from frozen cells (7 to 15 million of cells) stored in 1 ml of Tri Reagent RNA/DNA/protein isolation reagent (Molecular Research Center). The extraction was done by adding 200 µl of cold Ready Red-Chloroform isoamyl alcohol (MP Biomedicals, France) and vigorously shaking for 15 s using a vortex, then the sample was incubated on ice for at least 5 min. After centrifugation at 13 000 rpm for 15 min at 4°C, the upper aqueous phase was transferred into a new vial. One volume of isopropanol was added, the sample was vortexed and incubated for one hour at -20°C. After centrifugation at 13 000 rpm for 15 min at 4°C, the pellet was dried and 1 ml of cold 75% ethanol added. After centrifugation at 8000 rpm for 10 min at 4°C, the pellet was dried and incubated for 4 min at 65°C. The pellet was resuspended in 30 µl of RNAse-free water with RiboLock RNase Inhibitor (40U, Fermentas, France). RNA concentration was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc, Thermo Scientific, Brebières, France). RNA quality and purity was assessed on the Agilent 2100 BioAnalyser by using the Agilent RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Only RNA presenting a RIN (RNA Integrity Number) > 6.5 were selected for expression analysis (~ 90% of the samples had a RIN > 8). cDNA was generated from 1 µg of RNA with the SuperScript VILO cDNA Synthesis Kit (Invitrogen) for reverse transcription-PCR following the manufacturer’s suggestions. In order to ensure a good quality reverse transcription step, one part of each cDNA was used to check ABL1 (“TaqMan Gene Expression Assay”, Applied Biosystems, Hs01104728_m1) expression using ABI Prism 7300 HT (Applied Biosystems, Carlsbad, CA, USA). Specific target amplification and Quantitative PCR The other part of each cDNA was diluted in water to 5 ng/µl and used for target amplification by Biomark Dynamic Arrays (Fluidigm, BioMark, Pueblo, CO, USA). Inventoried TaqMan assays (Applied Biosystems) were pooled using 84 probes and primers pairs (72 DNA replication genes, 9 housekeeping genes - ABL, ACTB, B2MC, G6PD, GAPDH, GUSB, HPBS, TBP, UBC -, and BAALC, ERG, and MN1 genes) to a final concentration of 0.2X for each of the 84 assays. To increase sensitivity, a multiplexed pre-amplification process was performed for the pool on every 1.25 µl cDNA using 14 cycles cDNA pre-amplification step (95°C for 15s and 60°C 4 min) and Taqman PreAmp Master Mix (Applied Biosystems) in a standard PCR Thermocycler. Pre-amplified cDNA was diluted 1:5 in 10 mmol/L Tris, 1 mmol/L EDTA. Diluted cDNA (2.25 µl) was added to 2.5 µl Taqman Universal PCR Master Mix (Applied Biosystems) and 0.25 µl GE Sample Loading Reagent (Fluidigm). In a separate tube, 3.5 µl of Taqman Assay was added to 3.5 µl Sample Loading Reagent. 5 µl cDNA samples were loaded into the sample inlet wells, and 5 µl assay samples were loaded into assay detector inlets. Because 198 samples were to be analyzed in duplicates, five 96.96 Dynamic Arrays (Fluidigm) were used. For each plate, 1 well was loaded with H2O as a control for contamination. Genomic DNA (gDNA) from 3 different patients was loaded in order to check if TaqMan assays can also amplify gDNA. To verify STA (Specific Target Amplification) efficiency, a sample control gDNA and assay control RNAse P Taqman probe was treated (Lifetech PN 4316844), pre-amplified and quantified using the same master mix from the kit. The expected value cq (quantification cycle) was between 12 and 13. To perform inter-plate calibration, a sample calibrator made of cDNA from the patient #1 was included in duplicate in each plate. The chip was primed and placed into the NanoFlex Integrated fluidic circuit controller where 8 nl of cDNA and 1 nl of Assay were mixed. Real time PCR analysis was completed on the BioMark System (Fluidigm). The experiment was done on 2 series with 3 plates in the first serie and 2 plates in the second one. So to perform inter-plates calibration, a patient samples made of cDNA from patient #1 was used for the first serie and cDNA from samples #1, #30, #60, #90, #95 and #111 for the second one. The cDN1 froma patient sample #1 was also used as a between series control. Data processing Raw data obtained from the system’s software, using the auto detector function to establish the threshold setting (BioMark Realtime PCR Analysis V2.1.1, Fluidigm), were checked using the graphical representation of the plate layout. Among all reactions investigated, 0 were rejected due to bubbles or instable ROX (Carboxy-X-Rhodamin) signal. All amplification curves were displayed for each well of the calibrator sample. When the threshold for cycle did not meet quality criteria (that is the threshold occurred in the linear phase of the amplification curve instead of the exponential phase), the threshold value was set manually. The threshold established for the first Dynamic Array were applied to the 4 other Dynamic Arrays. Wells with very high (> 26), absent (999), or very low (< 2) endogenous Ct were excluded. Normalization method These raw data wre then normalized with the data obtained with the housekeeping genes. Then we performed the inter-plate calibration using the qbase+ algorithm as described (Hellemans, 2007). Among the 9 housekeeping genes tested [GUSB (Hs99999908_m1), ACTB (Hs99999903_m1), ABL1 (Hs01104728_m1), G6PD (Hs00166169_m1), TBP (Hs00427621_m1), GAPDH (Hs03929097_g1), HMBS (Hs00609293_g1), B2M (Hs00984230_m1), UBC (Hs00824723_m1)], GeNorm algorithm determined the four most stable, which were GAPDH, GUSB, TBP and ABL1, and were used to calculate the gene expression normalization factor. Expression values are given in CNRQ which corresponds to a value normalised and calibrated (2^ - deltaCT) (the reference value is 1, and every value can varie between 0 and the infinity). References: Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8(2):R19.
 
Contributor(s) David L, Fernandez-Vidal A, Bertoli S, Grgurevic S, Lepage B, Deshaies D, Prade N, Cartel M, Larrue C, Sarry J, Delabesse E, Cazaux C, Didier C, Récher C, Manenti S, Hoffmann J
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Submission date Sep 20, 2016
Last update date Sep 21, 2016
Contact name DAVID Laure
E-mail(s) laure.david@inserm.fr
Organization name Cancer Research Center of Toulouse
Street address 2 avenue Hubert CURIEN
City TOULOUSE
ZIP/Postal code 31 037
Country France
 
Platforms (1)
GPL22461 Fluidigm RT-PCR array for AML human samples
Samples (198)
GSM2322735 AML patient sample number 001 set1 plate 1
GSM2322736 AML patient sample number 002 set1 plate 1
GSM2322737 AML patient sample number 003 set1 plate 1
Relations
BioProject PRJNA343642

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE87128_Non-normalized_data.txt.gz 84.6 Kb (ftp)(http) TXT
Processed data included within Sample table

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