Transcription of マイクロアレイ実験 サンプル調整・データQC に関 …
1 QC 1. mRNA / miRNA / gDNA2. 3. QC GX9 1. - - - Agilent Invitrogen TRI zol Reagent(15596-026)Qiagen miRNeasy Mini kit(217004) Applied Biosystem mirVana RNA Isolation kit(AM1560) Total RNA smallRNA Qiagen DNeasyBlood & Tissue Kit(69504 69506)aCGH gDNAmiRNA Total RNA ULS aCGH FFPE DNA FFPE DNA UV A230 A260 1 = 40 ug /ml (RNA) 1 = 50 ug /ml (DNA) A280 A260/A280 = ~ : : A320 :230nm, 260nm, 280nm, 320nm RNA UV Max 260 nm 230 nm 260 RNA260 RNA260nm RNA DNA UV UV RNA DNA RNADNA gDNA Quant-iT PicoGreendsDNAA ssay Kit DNA RNA DNA DNA Total RNA Total RNA Total RNA 2.
2 3. Exchangeable cartridge for electrophoresis 16 pin electrodes Chip holder with heater plate16 pin electrodes connected to High Voltage sourceChip holder with heater plate1 (5 )2 12/11 (30 )3 Bioanalyzer Total RNA Bioanalyzer RNA IntegrityNumber Bioanalyzer Total RNA RIN .. RNA DegradedIntactRIN: 1 RIN: 2 RIN: 4 RIN: 3 RIN: 5 RIN: 6 RIN: 7 RIN: 9 RIN: 8 RIN: 10 Bioanalyzer Total RNA RIN .. RNA 10 RIN Total RNA -Electropherogram /rRNA /RIN 3 - RIN Total RNA 5 mRNA 3 gDNA ULS Labeling gDNA 10kb 10 7kb 5 7kb Agilent CGH ULS labelingprotocol gDNA ~ 5001221695 C agarose gel, 20 ng DNA, Sybr Gold stain ~ 5001221695 C 10 gDNA Enzymatic Labeling Direct WGA 500bp Agilent CGH Enzymatic labelingprotocol DNA (Female) HT29 Intact Intact gDNA 2.
3 -Wash - Agilent Wet Quality S/N Cy -Dye Cy5 Cy3 Branham et al.,BMC Biotechnology2007 Feb 12;7 :~2-4 ppb 100ppb GIS 2-M005 -01B 1760 710 1000mm TritonX-102 Wash Buffer 2 Agilent Agilent DNA 3. Agilent QC reportQC report 1 QC report 2 Up regulated Down regulated Feature (2color )Outlier Agilent Spike-In Mix( Option)-10 A RNA 6 - RNA Spike -In Mix 1color RNA Spike -In Mix 2color 1color spike-mix QC report 3 Spike -In Mix 1 QC GeneSpringGX9 Quality Control.
4 Agilent 1 Spike -In Control Spike -In Control Spike -In slope Non Uniformity Outlier Negative Control SD ..etc Agilent 1 Quality Control Metrics Quality Control Metrics Experiment Sample (PCA) Biological Replicates Agilent QC report Spike-In Control Thank you for your attention!
