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Guideline for Affymetrix Sample
Submission:
Services:
- 3’ Gene Expression Analysis-full
service
GeneChip
Human Genome U133 Plus 2.0, GeneChip Human Genome
U133A 2.0
GeneChip
Mouse Genome 430 2.0, GeneChip Mouse Genome
430A2.0 and GeneChip Rat Genome 230 2.0 are the
most arrays used for this service. The assay includes the following steps:
1). Check the quality and
integrity of the starting material- total RNA by using Agilent
2100 Bioanalyzer
2).
Reverse Transcription to synthesize first-strand cDNA
3). Synthesize second-strand cDNA
4). In
vitro Transcription to synthesize labeled aRNA
5). aRNA
purification and quantitation
6). Fragmentation of labeled aRNA and fragmentation reaction check by using Agilent 2100 Bioanalyzer
7).
Hybridization of the fragmented aRNA to the Genechip array
8). Probe array washing and staining
9). Probe array scanning and
generation of raw data by using Affymetrix
software and basic analysis of the microarray
data.
10) QC assessment of the array data
- Whole Transcript (WT) Expression Analysis-full
service
Human, mouse and rat Exon 1.0 ST Array
and Gene1.0 ST Array are arrays for this service. The assay includes
following steps:
1). Check the quality and
integrity of the starting material-total RNA by using Agilent
2100 Bioanalyzer
2). Synthesize first-strand cDNA
3). Synthesize second-strand cDNA
4).Synthesize cRNA using in vitro transcription
5).Purify cRNA
and assess cRNA yield
6).Synthesize 2nd-cycle
cDNA and hydrolyze using RNase
H
7). Purify 2nd-cycle
cDNA and assess cDNA
yield
8). Fragment the
single-stranded cDNA
9). Check the fragmentation by
using Agilent 2100 Bioanalyzer
10). Labeling of Fragmented
single-strand DNA
11). Target hybridization
12). Array washing, staining
and scanning
13). QC assessment of the array data
3. Cytogenetics
Copy Number Assay Service-full service
Genome-wide Human SNP Array 6.0 is used in this assay. The assay includes the following:
- Nsp ad Sty restriction enzyme digestion
- Nsp and Sty ligation
- Nsp and Sty PCR and PCR product check by running a
gel
- PCR
product purification
- PCR
product quantitation
- Fragmentation
and the fragmentation reaction check by running a gel
- Labeling
- Target
hybridization to genome-wide human SNP array 6.0
- Array
washing and staining
- Array
scanning and generation of raw data
- Assessment
of the quality of the array data and basic analysis
- Partial Microarray
Service
This service includes sample
hybridization, probe array washing, staining and scanning,
please refer to sample submission section below for details.
Sample Submission:
- Full Microarray
Services
Total RNA is the starting
material for 3’ Gene Expression Analysis and Whole Transcript (WT) Expression
Analysis. Genomic DNA is used as the starting material for Cytogenetics Copy Number Assay and gel image of gDNA which is assessed on 1% agarose
is required.
Please contact Xiaojun Zou (7-7776, zoux@georgetown.edu) for specific
requirements of each assay.
- Partial Microarray
Service
1) Quantification of
the cRNA: All RNA samples should meet assay
quality standards to ensure the highest quality RNA is hybridized to the
gene expression arrays. User should run the initial total RNA on an agarose gel and examine the ribosomal RNA bands.
Non-distinct ribosomal RNA bands indicate degradation. 260/280 absorbance
readings should be measured for both total RNA and biotinylated
cRNA. Acceptable 260/280 ratios fall in the range
of 1.8 to 2.1. Ratios below 1.8 indicate possible protein contamination.
Ratios above 2.1 indicate presence of degraded RNA, truncated cRNA transcripts, and/or excess free nucleotides.
2) Amount of the cRNA: 15 m g fragmented cRNA
is needed. The total volume is 40 m l (add RNase-free
water to adjust volume if necessary)
3) Please submit a copy
of the photograph of your fragment cRNA (plus
total RNA if possible) on the agarose gel
electrophoresis, and 260/280 absorbance reading(s) (biotinylated
cRNA only), together with the sample(s).
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