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| Status |
Public on Jun 15, 2016 |
| Title |
Plasma cfRNA from healthy subject1 |
| Sample type |
RNA |
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| Source name |
Plasma cfRNA from healthy subject
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| Organism |
Homo sapiens |
| Characteristics |
subject status/id: healthy subject1
gender: Female
age (yrs): 50
colonoscopy result: normal
molecule subtype: human plasma circulating free RNA
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| Treatment protocol |
Subject were classified as being either healthy, having adavnaced adenoma or cancer according to colonoscopy result or biopsy results.
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| Growth protocol |
not applicable
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| Extracted molecule |
total RNA |
| Extraction protocol |
Plasma total RNA was purified from 3.2ml of plasma mixed with 11.2 TRIzol reagent, with addition of 15 microgram of linear acrylamide. 200 μl of chloroform were added per each 1 ml of Trizol and mixed vigorously. After incubated for 10 minutes at room temperature, the mixture was centrifuged for 15 minutes at 14000 rpm at 4°C. The aqueous phase was transferred to a new tube and mixed vigorously with equal volume of chloroform, incubated for 3 minutes at room temperature and centrifuged for 15 minutes at 14,000 rpm at 4°C. Following the centrifugation, the upper phase was transferred to a new tube and mix thoroughly with a total of 2.8 ml of RLT plus buffer RNeasy mini kit (catalog no. 74104, Qiagen). This mixture was passed through the kit's gDNA eliminator mini spin column as described by the manufacture protocol. The flow-through was collected in a new tube, and 1.5 times volume of 100% EtOH was added. The solution was well mixed and incubated at -20°C for over-night. Next, 700 ml of the mixture was loaded on an RNeasy spin column and micro-centrifuged at 23°C, 14,000 RPM, for 30 seconds and flow-through was saved. The rest of the thawed solution was passed through the same RNeasy spin column, as described above.RNA purification process was completed by following the RNeasy mini kit protocol. The RNA-loaded RNeasy spin column were washed with 700 ml of RW1 buffer, centrifuged at 23°C 14,000 RPM for 30 seconds and flow through discarded. Additional two washes of spin column were done with 500 ml of RPE buffer. Finally, RNA was eluted in 35 ml of RNase-free water.
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| Label |
biotin
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| Label protocol |
For gene expression profiling using Affymetrix expression microarray, cDNA was synthesized using the Ovation PicoSL WTA System (catalog no. 3312, Nugen), according to kit's protocol. Subsequently, the cDNA was purified with MiniElute Reaction Cleanup kit (catalog no. 28204, Qiagen), according to kit protocol. Next, fragmentation and biotin labeling of the cDNA was done by Encore biotin module (catalog no. 4200, Nugen). The extent of fragmentation was monitored using Bioanalyzer 2100 Pico chip (Agilnet) using 1 µl of the reaction volume.
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| Hybridization protocol |
the use of the GeneChip® Hybridization, Wash and Stain Kit (catalog no. 900720, Affymetrix) and the GeneChip human 1.0 ST Arrays (catalog no. 901085, Affymetrix). Hybridization cocktail was prepared by mixing the following materials: 25 microliter of fragmented, biotin-labeled and amplified cDNA, 1.9 microliter control oligonucleotide B2 (3 nM), 5.5 microliter of 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 55 microliter 2X Hybridization Buffer, 11 microliter 100% DMSO, 11.6 microliter water. The procedure was performed according to manufacturer instructions.
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| Scan protocol |
Arrays were scanned on the Affymetrix GeneChip Scanner 3000 7G
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| Description |
BC12_NN
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| Data processing |
Raw CEL files were imported into Partek Genomics Suite and preprocessed using RMA background correction, quantile normalization and log2 transformation. No probe summarization to probeset was performed.
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| Submission date |
Jun 14, 2016 |
| Last update date |
Jun 15, 2016 |
| Contact name |
Vardit Moshayoff |
| Organization name |
BioMarCare technology
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| Street address |
Hadassaah Medical Center, Ein Kerem
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| City |
Jerusalem |
| ZIP/Postal code |
91100 |
| Country |
Israel |
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| Platform ID |
GPL10739 |
| Series (1) |
| GSE83353 |
Feasibility of unbiased RNA profiling of colorectal tumors: a proof of principle. |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM2200153_BC12_HuST.CEL.gz |
3.7 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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