title: sciTracer_Staphylococcus_aureus_N315_Scienion2002
status: Public on Mar 11 2013
contact.city: Bonn
contact.first: Peter
contact.last: Sass
contact.email: peter.sass@microbiology-bonn.de
contact.state: NRW
contact.address: Sigmund-Freud-Straße 25
contact.department: Institute for Microbiology (IMMIP)
contact.country: Germany
contact.institute: University of Bonn
organism: Staphylococcus aureus subsp. aureus N315
manufacture_protocol: The sciTRACER S. aureus contains 2338 PCR products from all 2593 ORFs in the annotated genome (Ref ID: NC_002745), arranged in unique and second sequences: since the ORFs sizes range from 51 bp (e.g.: 13700427) up to 2kb (e.g.: 13701232), additional second PCR products have been developed. As unique sequences, 2333 PCR products and as second sequences, 333 PCR products are immobilized from which 5 are not in the group of unique gene fragments. All unique products cover 90 % of all ORFs. All PCR fragments are purified, concentration normalized, dissolved in spotting solution and spotted robotically onto modified glass slides. They are represented twice (duplicates) on each array. Controls are usually spotted in the last row of each subarray block forming the control area and comprising controls for spiking and endogenous controls. The SCIENION control set consists of positive, negative and multifunctional elements. All references/controls are spotted in a special pattern enabling functional monitoring of the entire experimental process. The glass slides are processed and ready for hybridization reaction. For easy correlation of gene names to their spot position on the slide the “Spot Identification Pattern-Table” (gal-file) is available. Information source: The genes represented on sciTRACER S. aureus are selected from order requests derived from the University of Würzburg, Infectious Biology Group (Head: Prof. Dr. Jörg Hacker) at the Institut für Molekulare Infektionsbiologie. Generation of sequences: 1. The PCR fragments derive mostly from regions at the last 500 nt from the 3’ end and have an average size of 355 bps. In case that the predicted ORF displayed a size unsuitable for appropriate amplicon generation, the primer design was extended towards flanking regions in order to cover the desired ORF. 2. All selected sequences are designed to provide the highest specificity and display minimal similarity to other mRNA and DNA sequences within the specified host genome. 3. In order to obtain strong and comparable sensitivity and signal specificity, all fragments are designed to feature similar lengths, comparable G/C-contents and optimized melting temperatures. 4. Based on algorithmic and manual revisions of the predicted sequences, the designed PCR fragments are well suited for the generation of highly reliable gene expression data. Quality controls: All sequences are validated in silico and approved by size control and sequence verification. The spot morphology is checked by automated optical control and hybridization of labeled RNA or SyBRGreenI® assay. Functional tests are performed by complex hybridization with labeled reference RNA for each batch. Source Organism: Staphylococcus aureus N 315 (NC_002745, contig BA000018) Microarray mapping: Each SCIENION microarray consists of two regions: the hybridizing region and the identifier region. The identifier region carries a sticker showing the name of the product, the date of production, batch number and the ID of the array. This region also serves to grasp the slide. The DNA microarray is in the center of the hybridizing region. Spot distance and feature size: The spot distance (center to center) is 375 µm. The array size is approximately 54 mm x 18 mm (height x broadness). Each array consists of 48 blocks and each block comprises 12 x 11 spots. These blocks are separated by a blank line and display a defined order. to Product description: The sciTRACER S. aureus contains 2338 PCR products from all 2593 ORFs in the annotated genome (Ref ID: NC_002745), arranged in unique and second sequences: since the ORFs sizes range from 51 bp (e.g.: 13700427) up to 2kb (e.g.: 13701232), additional second PCR products have been developed. As unique sequences, 2333 PCR products and as second sequences, 333 PCR products are immobilized from which 5 are not in the group of unique gene fragments. All unique products cover 90 % of all ORFs. All PCR fragments are purified, concentration normalized, dissolved in spotting solution and spotted robotically onto modified glass slides. They are represented twice (duplicates) on each array. Controls are usually spotted in the last row of each subarray block forming the control area and comprising controls for spiking and endogenous controls. The SCIENION control set consists of positive, negative and multifunctional elements. All references/controls are spotted in a special pattern enabling functional monitoring of the entire experimental process. The glass slides are processed and ready for hybridization reaction. For easy correlation of gene names to their spot position on the slide the “Spot Identification Pattern-Table” (gal-file) is available. Information source: The genes represented on sciTRACER S. aureus are selected from order requests derived from the University of Würzburg, Infectious Biology Group (Head: Prof. Dr. Jörg Hacker) at the Institut für Molekulare Infektionsbiologie. Generation of sequences: 1. The PCR fragments derive mostly from regions at the last 500 nt from the 3’ end and have an average size of 355 bps. In case that the predicted ORF displayed a size unsuitable for appropriate amplicon generation, the primer design was extended towards flanking regions in order to cover the desired ORF. 2. All selected sequences are designed to provide the highest specificity and display minimal similarity to other mRNA and DNA sequences within the specified host genome. 3. In order to obtain strong and comparable sensitivity and signal specificity, all fragments are designed to feature similar lengths, comparable G/C-contents and optimized melting temperatures. 4. Based on algorithmic and manual revisions of the predicted sequences, the designed PCR fragments are well suited for the generation of highly reliable gene expression data. Quality controls: All sequences are validated in silico and approved by size control and sequence verification. The spot morphology is checked by automated optical control and hybridization of labeled RNA or SyBRGreenI® assay. Functional tests are performed by complex hybridization with labeled reference RNA for each batch. Source Organism: Staphylococcus aureus N 315 (NC_002745, contig BA000018) Microarray mapping: Each SCIENION microarray consists of two regions: the hybridizing region and the identifier region. The identifier region carries a sticker showing the name of the product, the date of production, batch number and the ID of the array. This region also serves to grasp the slide. The DNA microarray is in the center of the hybridizing region. Spot distance and feature size: The spot distance (center to center) is 375 µm. The array size is approximately 54 mm x 18 mm (height x broadness). Each array consists of 48 blocks and each block comprises 12 x 11 spots. These blocks are separated by a blank line and display a defined order.