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.
2 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. 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.
3 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.
4 -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.
5 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.
6 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!
