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Nucleic acid Amplification Service
Laser Capture Microdissection Service
Microarray Service
Bioinformatics Service
Real time PCR service

Microarray service
Welgene Biotech has implemented the complete Agilent microarray platform to ensure the best array experiment quality. The array service quality has certified by ISO17025 and Agilent certified service provider program. The whole system includes:


¡E sample QC facilitated by the 2100 bioanalyzer
¡E sample labeling and hybridization using reagents and protocol suggested by Agilent
¡E microarray analysis and array image QC on the Agilent microarray scanner using the Feature Extraction Software.


Currently we provide the array service including¡G
Gene expression microarray
Array CGH
ChIP-on-chip

MiRNA

Gene expression ¡G
The arrays we offer are Agilent oligo microarray. With SurePrint technology, Agilent¡¦s 60-mer oligo format allows for rapid design changes so researchers can more freely iterate microarray design and tailor it to experimental needs.

The probe design of Agilent array considers the Tm matching, secondary structure of probes, sequence specificity, and fully validated by experiments. Recently, MAOC report published in the Nature Biotechnology September 2006 demonstrated that the correlation of array data of Agilent¡¦s platform with Taqman assay is over 0.9. Research publications conducting Agilent microarray so far have over hundreds of papers, the detail information could be browsed in the OpenGenomics website.

Array choice

 whole human genome whole mouse genome whole rat genome whole rice genome  Arabidopsis 3
 
array format		 4 x 44K 4 x 44K 4 x 44K 4 x 44K 4 x 44K
features/ array 45,015 45,018 45,018 ~45,000 45,018
genes coverage ~41,000 ~41,000 ~41,000 ~40,000 ~40,000
Content source Goldenpath, Ensembl, Unigene, Human Genome (Build 33), Refseq, GebBank USC mRNA known genes, Natl. Institute on Aging, Genbank, Unigene, Refseq, Ensembl, RIKEN Ensembl, UCSC Goldenpath, Unigene, Refseq, Genbank Japan's Natl. Institute of Agrobiological Science KOME library database TIGR ATH1 v.5 database, RefSeq, GenBank, Univ. of Delaware's Arabidopsis MPSS database
Design ID 014850 014868 014879 015241 015059
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For the arrays of other species, please access Agilent websit, we can order it for you.

Sample type: total RNA
RNA amount RNA concentration OD260/280 28S/18S Ratio RIN
5 µg ¡Ù 0.2 µg / m l ¡Ù 1.8 ¡Ù 1.2 ¡Ù 7
*If the specimens provided were cell lines or tissues, please access our Nucleic acids purification service for more detail information.
** For customers of overseas would like to ship RNA, please fill-in the Custom Invoice, and fax to 886-2-26557867, attention to Stevano Tsia..

Description of RNA QC:
1. Measurement of quantity and purity: To save the customers¡¦ specimen, we have used highly sensitive spectrophotometer- ND1000 (NanoDrop) to measure the quantity (OD 260nm) and purity (260/280 ratio), the sensitivity is ~5 ng, and takes only 1ul of the sample.
2. Measurement of RNA integrity: The RNA integrity is measured by RNA 6000 nanochip run by 2100 bioanalyzer. The quality is digitalized by providing the 28S/18S ratio and RIN (RNA Integrity Number)
3. RNA QC suggested¡G
OD260/280 28S/18S Ratio RIN
¡Ù 1.8 ¡Ù 1.2 ¡Ù 7

Workflow of the gene expression microarray experiments
Service workflow Description
a pair of total RNA (experiment vs. control) provided by customers
RNA QC by OD and 2100 bioanalyzer
cRNA synthesis and labeling reaction
hybridization and wash
image analysis and data output

Service report of gene expression microarray

 

Report output   Description

  Format
Content
(images)
 Scanning image Combined Cy3,Cy5 image TIFF, JEPG
Scatter plot Distribution of the intensity along the X-¡BY-axis JEPG
MA plot Distribution of the M and A along the Y-¡BX-axis JEPG
Content
(data)
 Raw data Original data output by Feature Extraction Excel
Normalized ratio (normalized by rank consistent lowess method) Statistics analysis Excel
p-value(with Agilent¡¦s error model to distinguish the significant difference between two channels) Statistics analysis Excel
Significant up & down regulated gene list Fold change ¡Ù2 ¡A and P-value< 0.05 Excel

array CGH¡G
The arrays we offer are Agilent oligo microarray. With their SurePrint technology, Agilent¡¦s 60-mer oligo format provides the most sensitive platform to allow the detection of chromosome aberrations.

The probe design of Agilent array considers the Tm matching, secondary structure of probes, sequence specificity, The performance of Agilent aCGH array also has been published in PNAS. As the data shown below, aCGH result of GM50122 18q- cell line indicated a single copy deletion at 5.945~5.955 which is correlated with the previously known locus at 59,462,941.
Research publications conducting Agilent aCGH microarray so far have over tens of papers, the detail information could be browsed in the OpenGenomics website.
Currently we provide aCGH arrays and their specifications are listed as followed:
Agilent aCGH array type Human 105A Human 244A Mouse 105A Mouse 244A
Product number 4412A 4411B 4416A 4415A
Feature size 65 µm 65 µm 65 µm 65 µm
Resolution 15 kb, 105K 6.4 kb, 244K 15 kb 6.4 kb
% of intergenic probe 71 70 69.6 68.4
% of intragenic probe 29 30 30.4 31.6
Probes per gene ¡Ù2 (96%) ¡Ù3 (99%)
¡Ù 4 (96%) 		¡Ù2 (97.5%) ¡Ù3 (99%)
¡Ù4 (97.4%)
Other regions covered miRNA, promoter, telomere miRNA, promoter, telomere miRNA, promoter, telomere miRNA, promoter, telomere
# biological features (x3) 525 1,000 760 1,400
# control features 4,626 5,045 4,712 5,099
Content Source UCSC hg17 (NCBI Build 35) UCSC hg17 (NCBI Build 35) UCSC mm7 (NCBI Build 35) UCSC mm7 (NCBI Build 35)

Sample type: total DNA or genomic DNA

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DNA amount

DNA conc.

OD260/280

DNA

4 µg ¡Ù100ng/ µl ¡Ù1.7

Low amount of DNA
(DNA amplification by phi29 polymerase)

 0.5 µg ¡Ù50ng/ µl ¡Ù1.7
*If the specimens provided were cell lines, tissues or blood cells, please access our Nucleic acids purification service for more detail information.

Description of DNA QC:
1. Measurement of quantity and purity: To save the customers¡¦ specimen, we have used highly sensitive spectrophotometer- ND1000 (NanoDrop) to measure the quantity (OD 260nm) and purity (260/280 ratio), the sensitivity is ~5 ng, and takes only 1 ul of the sample.
2. Measurement of DNA integrity: The ssDNA integrity is measured by RNA 6000 nanochip run by 2100 bioanalyzer.
3. DNA QC suggested¡G
OD260/280
¡Ù 1.8

Workflow of the aCGH microarray experiments
Workflow Description
a pair of total DNA (experiment vs. control?) provided by customers.
RNA QC by OD and 2100 bioanalyzer
experiment and reference DNA were random priming labeled with Cyedye-dUTP
hybridization and wash
image analysis and data output
If customers do not have appropriate control, we can provide Promega¡¦s normal male DNA (cat.# G1471) and normal female DNA (cat.# G1521) as the reference.

Service report of gene expression aCGH

 

Report output   Description

  Format
Content
(images)
 Scanning image Combined Cy3,Cy5 image TIFF, JEPG
Chromosome view Scatter plot of aberrations of each chromosome JEPG
Content
(data)
 Raw data Original data output by feature extraction txt
Gene list involved in the chromosome aberration Gene list Excel
Normalized ratio (by linear normalization) Statistics analysis Excel

ChIP-on-chip (chromatin immunoprecipitation-on-chip) ¡G
The arrays we offer are Agilent oligo microarray. With their SurePrint technology, Agilent¡¦s 60-mer oligo format provides the most sensitive platform to allow the detection of chromosome aberrations.

The probe design of Agilent array considers the Tm matching, secondary structure of probes, sequence specificity. Agilent ChIP-on-chip arrays has been applied in the research of chromatin modulations, developmental regulation of stem cells, and DNA repair process, etc.
Research publications conducting Agilent Chip-on-chip microarray have been published in the journals such as Nature¡AScience¡ACell etc., and the detail information could be browsed in the OpenGenomics website.

Currently we provide aCGH arrays and the specifications are listed as followed:
Agilent type Array format Target Probe resolution
Yeast 244K x 1 Contains ~85% of the non-repetitive portion of the yeast genome(~12 MB) ~50 nt
Human Promoter 244K x 2 ~17,000 of the best-defined human transcripts represented as defined by RefSeq
Content source from UCSC hg17/NCBI 		Set covers -5.5kb upstream to +2.5KB downstream of the transcriptional start sites
~25 probes/gene
Spacing ~195 nt
Mouse Promoter 244K x 2 ~17,000 of the best-defined mouse transcripts represented as defined by RefSeq
Content source from UCSC mm7/NCBI release 35(August 2005)		 Set covers -5.5kb upstream to +2.5KB downstream of the transcriptional start sites
~ 25 probes/gene
Spacing ~200 nt
Human CpG Islands 244K x 1 Content source from UCSC hg17/NCBI
27,800 CpG islands 		237,220 probes in or within 95bp of CpG Islands
195K CpG probes, 50K non-CpG
Drosophila 244K x 2 Enriched content source from UCSC dm2 Tiled across whole Drosophila genome with ~233 nt
Arabidopsis 244K x 2 Content source from UCSC ath1 Tiled across whole Arabidopsis genome with~ 212 nt
C. elegans 244K x 2 Content sourced from UCSC ce2 Tiled across whole C. elegans genome with ~182 nt

For the arrays of other species, please access Agilent websit, we can order it for you.

Workflow of the ChIP-on-chip microarray experiments
Workflow Descriptions
Please download the detail SOP for chromatin-IP experiment
Customer should prepare hen LM-PCR products from the chromatin-immunoprecipitation

preparation of 5x10 7 ~ 1x108 cells
formaldehyde treatment to crosslink chromatin complex

Bind the antibody to the magnetic beads

lyze the cells, and fragment DNA to 100bp-500bp

Immunoprecipitate the chromatin
reverse cross-link, and purify the DNA

QC with promoter-specific primer to check the successfulness of Ch-IP
Shipment of IP- and Input-DNA to Welgene¡¦s service lab

RNA QC by OD and 2100 bioanalyzer

QC with promoter-specific primer to check the successfulness of LM-PCR

Blunt the DNA ends and ligate the linkers
LM-PCR to amplify the IP and Input sample

random primingto label the ChIP with Input-DNA

hybridization and wash

image analysis and data output

DNA required for the analysis are suggested as followed:
ChIP DNA , ~3µg, length profile at 100~500 bp

Input DNA, ~ 3µg, length profile at 100~500 bp

Promoter specific primer , 5µm, 20µl

Service report of ChIP-on-chip

 

Report output   Description

  Format
Content
(images)
 Scanning image Combined Cy3,Cy5 image TIFF, JEPG
Scatter plot Distribution of the intensity along the X-¡BY-axis JEPG
MA plot Distribution of the M and A along the Y-¡BX-axis JEPG
Content
(data)
 Raw data Original data output by Feature Extraction Excel
Normalized ratio ( by  LOWESS normalization) Statistics analysis Excel
Identify the binding event by error model Statistics analysis Excel

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microRNA array ¡G
The arrays we offer are Agilent oligo microarray. With their SurePrint technology, Agilent¡¦s 60-mer oligo format provides the most sensitive platform to allow the detection of chromosome aberrations.

The probe design of Agilent array considers the Tm matching, probe specificity to reduce the cross hybridization of longer RNA such as rRNA, mRNA amd pre-miRNA. The performance has been validated and the relative result has been published at RNA journal.
RNA sample applied to Agilent miRNA array only takes 100ng total RNA without any size-fractionation steps.
Research publications conducting Agilent miRNA microarray so far have over tens of papers, the detail information could be browsed in the OpenGenomics website.
Agilent microRNA available Human  v1 human v2 mouse rat
Product number G4470A G4470B G4472A G4473A
Feature size 65 µm 65 µm 65 µm 65 µm
Probe length 60-mer 60-mer 60-mer 60-mer
Total feature ~15,000 ~15,000 ~15,000 ~15,000
total  miRNA gene no 534 799 577 350
Total human/ mouse/ rat miRNA gene no. 470 723* 567* 350*
Total human viral miRNA gene no. 64 76 10¡@ 0
Replicate features per miRNA 20-40 20-40 20-40 20-40
Content Source Sanger miRBase Release 9.1 Sanger miRBase Release 10.1 Sanger miRBase Release 10.1 Sanger miRBase Release 10.1
Labeling type

Direct end labeling using Cyanine 3 pCp
The miRNA gene no. registered on miBase 10.1 is 541 for human, 443 for mouse, and 287 for rat, the extra genes designed in the Agilent array are for the sequence variants.


RNA required for the analysis are suggested as followed:

¡@

RNA amount

RNA conc.

OD260/280

total RNA

0.5µg ¡Ù100ng/ µl ¡Ù1.7

*If the specimens provided were cell lines or tissues, please access our Nucleic acids purification service for more detail information.
** For customers of overseas would like to ship RNA, please fill-in the Custom Invoice, and fax to 886-2-26557867, attention to Stevano Tsia.

*** Current we suggest RNA isolation by Trizol reagent (Invitrogen #15596-018)).

¡@


Description of RNA QC:
1. Measurement of quantity and purity: To save the customers¡¦ specimen, we have used highly sensitive spectrophotometer- ND1000 (NanoDrop) to measure the quantity (OD 260nm) and purity (260/280 ratio), the sensitivity is ~5 ng, and takes only 1ul of the sample.
2. Measurement of RNA integrity: The RNA integrity is measured by RNA 6000 nanochip run by 2100 bioanalyzer. The quality is digitalized by providing the 28S/18S ratio and RIN (RNA Integrity Number)
3. RNA QC suggested¡G
OD260/280 28S/18S Ratio RIN
¡Ù 1.8 ¡Ù 1.2 ¡Ù 6


Workflow of the miRNA microarray experiments

Workflow Description
0.5 mg of total RNA provided by user
  QC by OD260/280 ¤Î 2100 Bionanalyzer QC
end labeling of miRNA by T4 RNA ligase
  hybridization and wash
array image analysis and dataoutput
¡@


Service report of miRNA microarray

 

Report output   Description

  Format
Content

(Image)

 Scanning image  Cy3,mage TIFF, JEPG
Content

(data)

 Raw data Original data output by Feature Extraction Excel
Ratio ( without normalization)¡¯ Statistics analysis Excel

* So far no true ¡§housekeeping¡¨ miRNAs have been demonstrated to have constant expression across samples, and other RNA species looked to as surrogates for small RNAs (tRNAs, snRNAs, 5S rRNA, etc) are also variable. Ideally, the miRNA profiles generated on the Agilent platform are normalized to the amount of total RNA (i.e. input 100ng), since equal amounts of total RNA are used for each assay.

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