Affymetrix Gene Expression Service Lab Report

Transcription

1 Report file for #1 1 st QC of RNA quality Affymetrix Gene Expression Service Lab Report Assigned no Number we assigned Threshold User User name N/A User Sample ID Sample name that user give N/A Sample type RNA type N/A Submitted Date Date of sample submission N/A UV spectrophotometer (0.7 µl RNA µl 10mM Tris ph 8.0 buffer) 230 value of OD ~ value of OD ~ value of OD ~ value of OD 320, background ~ 3 260/230 ratio OD / OD > = / 280 ratio OD / OD > = 1.9 Concentration (µg/µl) > = 1.5 Agilent Bioanalyzer (following three data come from Bioanalyzer) 28S peak > 18S peak It dependents on different species 28S peak > 18S peak 28S / 18S ratio The ratio of 28S/18S > = 1.4 total area of (28S% + 18S %) Total area of 28S% + 18S% > = 40 1 of 7

2 2 nd and 3 rd : QC for the efficiency of IVT and Fragmentation IVT (In vitro transcription) Date Date of IVT reaction N/A IVT kit 3 - Amplification for IVT Labeling N/A 230 OD 230 of crna ~ OD 260 of crna ~ OD 280 of crna ~ OD 320 of crna, background ~ 3 260/230 ratio OD / OD > /280 ratio OD / OD >1.9 Concentration (µg/µl) conc. of crna > = 1.5 Gel Fragmentation Gel crna was smear from 0.3 kb to 3kb (DNA ladder) on 1% agarose gel. crna fragmentation from 100 bp to 300 bp (DNA ladder) on 1% agarose gel. Btwn 0.3 ~ 3 kb Btwn bp 2 of 7

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4 Hybridization, wash/stain and image processing record Chip Hybridization Date Date of hybridization N/A Chip type Chip type of usage N/A Lot# GeneChip lot no. GeneChips from the same lot are used for one user if possible N/A Hybridization hours 16.5 hr N/A Wash / Stain Date Date of washing / staining N/A Protocol Wash/stain protocol of Fluidic Station 450 N/A Image Data (data analysis and produce after chip scanning) Date date of image scanning N/A Grid aligned Aligned to four corners of chip OK Borders stained Staining of the border of chip for alignment OK 4 th QC of hybridization control Report file Sample assigned no. N/A Scaling factor TGT value For eliminating the non-biological effect between GeneChips, it is necessary for signal normalization before data comparison. Scaling factor standardizes the average signal of each experiment to a TGT value. If the scaling factor with the same TGT value of compared GeneChip differs by more than 3-fold, it should be treated with caution. Default value was 500, please see scaling factor N/A Def = 500 Background Value of chip background <100 Percentage of Present The percentage of significantly expressed probe sets > 30 4 of 7

5 AFFX-Actin 3 / 5 ratio AFFX-GAPDH 3 / 5 ratio AFFX-r2-BIOB AFFX-r2-BIOC AFFX-r2-BIOD AFFX-r2-CRE The 3 /5 ratio of housekeeping gene reveals the IVT efficiency. The 3 /5 ratio of housekeeping gene reveals the IVT efficiency.. < 3, approx 1 < 3, approx 1 P or A P P P 5 of 7

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7 Summary of the QC report file 1. QC result All of QC results are fine. 2. TGT value and the Scaling factor The default of TGT value is 500. Please take care of the value of TGT value when doing comparison. And if the scaling factor is greater than 3-fold of one another, please treat the data with caution. 3. Number Present It depends on your species, treatments, etc. 4. Housekeeping Control The Sig (3 /5 ) of AFFX-Actin and AFFX-GAPDH should not over than 3. The high value means the inefficiency of the IVT reaction. 5. Spike Controls Please note that the Det (5 ) and Det (3 ) of AFFX-BIOC, AFFX-BIOD and AFFX-CRE probe sets must be P (Present). And the AFFX-BIOB might be P or A (Absent), it is because the concentration we spike in is the limitation of the detection. Affymetrix Gene Expression Service Lab affy@gate.sinica.edu.tw ext of 7