Empire Genomics | Frequently Asked Questions

The labeling kit we would recommend for your probes would be as follows:

Nick Translation Kit 50 Reactions
From Abbott Molecular
SKU: 07J00-001

For DNA purification we recommend this kit:

Agencourt CosMCPrep - High/Low Copy Fosmid and BAC Purification
Item No: A29174

You can find the Material Saftey Data Sheet for Labeled BAC clones by clicking the following link: Material_Safety_Data_Sheet.pdf

It is possible that when your package arrives, all of the dry ice will have evaporated. Our clones are stable at room temperature and are therefore still useable.

Yes. We can create 2, 3 and 4 probe cocktail panels upon request. Pricing is dependent upon probe volume and dyes used. For more information please contact us at:

701 Ellicott Street
Suite 203
Buffalo, New York 14203
USA
info@empiregenomics.com
1-800-715-5880
1-716-849-6890

Repeat the assay on a new specimen using one of the following:
- Decrease the melt temperature 2°C
- Decrease the melt time until morphology becomes adequate

Pretreat the slides
1. Prepare a solution of 2X SSC/1% paraformaldehyde
2. Immerse the slide for 1 minute in 2X SSC/1% paraformaldehyde
3. Immerse the slide in purified water several times
4. Dehydrate slides through 1 minute series of ethanol rinses (70%, 85%, 100%)
5. Air dry the slides and continue preparing slide for Codenaturation

If poor morphology continues, modify use of denaturation/hybridization system

Prepare 280 uL 70% formamide/2X SSC denaturation solution (196 uL formamide/28 uL 2X SSC/56 uL purified water
Run HYBrite or ThermoBrite Hold Temp program set at 73°C
Place 10uL of 70% formamide/2X SSC denaturation solution on each target area and cover slip
Upon HyBrite or ThermoBrite reaching 73°C, place slides on heating surface and close the cover
After 3 minutes, remove the slides
Remove the cover slip
Continue in dehydration step of non-codenaturation assay process

Repeat hybridization using one of the following:
- Decrease the melt temperature by 2?C
- Decrease the melt time until signal intensity becomes acceptable

Repeat the assay on a new specimen using one of the following:
- Increase the hybridization time
- Increase the melt temperature as needed until the signal becomes adequate
- Wash the slide with 0.4X SSC/0.3% NP-40 at 70-73 ?C

Repeat the assay on a new specimen using on of the following:
- Increase the temperature of 0.4X SSC/0.3% NP-40 by 2?C; continue to increase the temperature as needed
- Decrease the melt temperature by 2?C

Did the counterstain appear too weak?
-Remove cover slip and Immerse slides in 2X SSC/0.1% NP-40 at ambient temperature for 5 minutes; agitate occasionally. -Dehydrate slide through series of ethanol rinses. Air dry and reapply new counterstain

Was the wrong concentration of counterstain used?
-If too bright, dilute the counterstain in antifade solution before applying

Was the counterstain too old or exposed to light for an extended period?
-Store counterstain at -20?C, protected from light
-Do not use counterstain past its expiration date

Were the probes diluted inappropriately?
-Ensure the probe mixture was made according to protocol specifications

Were the conditions inappropriate for hybridization?
-Ensure incubator is at the proper temperature of 37?C
-Check that the hybridization buffer was added to the probe mixture and in the adequate amount

Was the wash temperature too low?
-To maintain the wash temperature, place no more than four slides in the wash at a time and ensure temperature of the wash solution is correct prior to washing another set

Was the wash solution stringency too low?
-Ensure wash solutions are made according to protocol
-The lower the concentration of salt, the higher the concentration of formamide and NP-40, and subsequently, the more stringent the wash

Was the specimen slide not adequately denatured?
-Ensure temperature of denaturation solution is 731?C prior to immersing slide
-Increase temperature of denaturation solution to 74?
-Increase the time slide is immersed in denaturation solution by 2-4 minutes

Was the specimen slide not adequately prepared for FISH?
-Contact Empire Genomics Technical Support
-Refer to FISH probe protocol

Were the specimen slides improperly aged after dropping the specimen?
-Age slides at ambient temperature for 24 hours prior to FISH

Were the specimen slides not dried thoroughly enough prior to immersion?
-Dehydrate slides through a series of 1 minute ethanol rinses (70%, 80%, and 100%)

Was specimen GTG-banded?
-Prepare fresh specimen slides
-Using trypsin-Giemsa banded specimens for FISH may require adjustments in banding and/or hybridization protocol- For more information contact Empire Genomics Technical Support

Was the probe not added?
-Prepare a new probe mixture, allowing enough time for probe to thaw. Vortex or pipette reagents to mix; centrifuge briefly. Pipette probe slowly

Was one or all of the components (probe, hybridization buffer, probe mixture) not mixed well prior to use?
-Use pipet to mix or vortex reagents; briefly centrifuge

Were the probes not properly diluted for hybridization?
-Maintain proper ratio of probe mix by using the volumes stated in the probe preparation procedure add 7 L hybridization buffer, 1 L CEN probe to 2 L of probe
-Ensure the pipette is calibrated. Allow hybridization buffer to thaw completely and reach ambient temperature prior to use; pipette slowly

Was the probe not adequately denatured?
-Ensure temperature of water bath used to denature the probe mix is 731?C
-Denature probe mixture for 5 minutes
-Plan so the probe is applied directly after removal of slides from the 100% ethanol solution (ensure ethanol has completely evaporated prior to application)
-Remove tube containing probe mix from the water bath and place immediately onto 45-50?C slide warmer.
-Keep tube on warmer while pipetting the probe onto the slide
-Note: only process as many slides as you can while maintaining proper temperatures and times outlined

Was the probe mix dried too thoroughly on specimen slide?
-Immediately place the cover slip over the target area after application of probe mix
-Washing the hybridization, remove cover slips from slides one at a time. Immerse each slide immediately into wash solution upon removal of slip before removing the next

Did you see air bubbles trapped under the cover slip during hybridization?
-In applying cover slip, first touch the surface of the probe mixture. Place the slide with cover slip down on a blotter, gently press out visible bubbles

Were the hybridization conditions not optimal?
-Ensure that the stated time and temperatures in protocol are strictly followed
-Ensure temperature of the incubator is 37?C
-Seal cover slip with rubber cement to ensure there are no gaps
-Increase the hybridization time

Were the wash conditions or solutions used incorrect?
-Adhere to protocol in making wash solutions and setting temperatures
-Ensure the thermometers and pH meters used are calibrated properly
-Before immersing slides in wash solution, remove cover slips

Were the probes or specimen slides stored improperly?
-Store undiluted probe at -20?C in the dark
-Store non-hybridized slides desiccated at -20?C (for extended periods) or at ambient temperature (for shorter periods)
-Store hybridized slides at -20?C in dark for up to 6 months

Was the wrong filter set used to view hybridization?
-Probe signals may appear fainter if viewed through multi-bandpass set; use correct filter for flourophores

Was the microscope configuration or objective not adequate to view FISH results? Are the filters damaged?
-Contact your microscope manufacturer

Was slide cleaned properly prior to sample preparation?
-Immerse slide in ethanol, dry using lint-free paper

Were the Specimen slides used too soon prior to denaturation?
-Ensure slides are aged at least 24 hours at ambient temperature

Could there have been too much cellular debris on the sample?
-Wash cell pellet with fresh fixative three times prior to dropping slides

Were the metaphase spreads aged by baking, or do they contain cytoplasm?
-Increase time the slide is immersed in the denaturation solution to 10 minutes

Was the slide inadequately washed following hybridization?
-Ensure wash solutions were made according to procedures outlined by the package insert
-Make sure the wash solution is at the proper PH and temperature, remove cover slip and repeat the wash procedure
-Increase immerse time to 4 minutes in the 73?C 0.4XSSC/0.3% NP-40 wash

Were the wash solutions used too long or improperly stored?
-Ensure wash solutions containing formamide are properly stored at 4?C
-Discard these solutions after 7 days or after frequent use; discard all other solutions after 1 day
-Ensure the pH of formamide solutions are pH 7.0-8.0

Was the hybridization viewed using long bandpass filters?
- Reduce background light by switching to filters with smaller bandwidths or to multi-bandpass filters

Did the slides dry too quickly during preparation?
-Increase the humidity used when dropping slides, or increase the temperature of the water bath.
-Decrease slide warmer temperature during preparation of samples
-Let slides dry overnight; age for at least 24 hours at room temperature
-Do not bake slides at high temperature

Was the specimen over-denatured?
-Adhere to directions on package insert to make denaturation solution
-Prior to immersing the slide, ensure temperature of the solution is at 731?C, decrease temperature to 72?C
-Decrease the time of slide denaturation process by 1-3 minutes

This is how other vendors' dyes compare to EG:

Color	Label Description	Ex. Max	Em. Max
Aqua	DEAC-dUTP	433 nm	480 nm
Green	5-Fluorescein dUTP	497 nm	524 nm
Green	Cyanine-3 dUTP	509 nm	538 nm
Gold	5(6)-Carboxyrhodamine 6G dUTP	530 nm	555 nm
Orange	5-TAMRA dUTP	559 nm	588 nm
Red	5-ROX dUTP	592 nm	612 nm
Red	Cyanine-5 dUTP	655 nm	675 nm

We provide a 2 year guarantee on all FISH probes from the date of manufacturing.

The following dyes are available when ordering our Custom Labeled BAC Probes:

Color	Label Description	Ex. Max	Em. Max	Ext. Coeff.
Aqua	DEAC-dUTP	431 nm	480 nm	50000
Green	5-Fluorescein dUTP	496 nm	520 nm	85000
Gold	5(6)-Carboxyrhodamine 6G dUTP	525 nm	551 nm	92000
Orange	5-TAMRA dUTP	552 nm	576 nm	60000
Red	5-ROX dUTP	580 nm	603 nm	75000

Yes. Please download the PDF version of our Labeled FISH Probe Instruction Guide. If you have any additional questions, please feel free to contact us at info@empiregenomics.com.

Each labeled FISH probe order enables you to perform 5 hybridization experiments. For more information, please review the Labeled FISH Probe Instruction Guide.

If you already know the clone name (ie: RP11-79G24) of the BAC you would like to have fluorescently labeled, please go directly to our labeledFISH probe ordering page.If you would like to search for a clonebased on a gene or chromosomal region of interest, visitCloneCentral- our new BAC clone search utility.

You can receive your order in as quick as 7 days. Please note: Orders are currently processed once a week and must be received by Monday at 12 noon EST for quickest turnaround time. Average turnaround time is 10-14 days.

All labeled BAC probe orders comein a 5-test kit format which includesa total quantity of1ug of DNA.Click hereto learn more about our Custom Labeled FISH probes.

You can find the Material Saftey Data Sheet for BAC clones by clicking the following link: Material_Safety_Data_Sheet.pdf

You can store your unlabeled BAC clones in a standard 4 degree freezer.

In the course of our initial work with PAC libraries and PAC vectors, we obtained feedback from several sequencing centers who considered the costs of sequencing PAC versus BAC clones. A disadvantage was the size of the PAC vector (16 kb left in recombinants) as compared to the size of the BAC vector (7 to 8 kb, depending on the vector variant). For the use of BACs versus PACs with inserts in the range of 100-150 kb, this means that a larger fraction of the clones in shotgun sequence libraries (M13 or pUC) represents the PAC/BAC vector sequence. This results in a few percent increase in the cost per base pair of sequence reads.

We have, therefore,improved the BAC vector (Shizuya H. et. al. 1992, Proc. Natl. Acad. Sci. USA 89:8794-8797) to include some of the retrofitting options included in our pPAC4 vector We also included the sacBII gene for use as a positive-selection marker to favor recombinant clones over non-insert background colonies. The sacBII gene from Bacillus amyloliquefaciens was derived from Nat Sternberg's P1 vector (Pierce et al. 1992, Proc. Natl. Acad. Sci. USA 89:2056-2060). The toxicity of the sacBII gene is a result of the conversion of fructose (derived from sacharose) into polyfructose (levan), which is toxic in E.coli. The cloning vector and recombinant clones do NOT express the sacBII gene and hence are sucrose-resistant. Undesirable clones derived from the deleted cloning vector (without the "green" stuffer fragment) are sucrose-sensitive and ,hence, do not form colonies on sucrose-containing media.

The new vector has been named pBACe3.6. Specifically, we maintained the wildtype loxP site, added an additional mutant loxP511 site, a site for the intron encoded nuclease PI-SceI and the Tn7att site.

The presence of this pUC plasmid serves dual functions: high copy number of the vector for preparing large quantities and appropriate disruption of the sacBII gene to increase viability of the vector containing strain. In addition to the BamHI site, 5 additional sites can be used for preparing BAC libraries: SacI, SacII, MluI, EcoRI, and AvaIII. The EcoRI site is particularly important in view of the use of this site in the RARE cleavage procedure, thus opening the possibility for selective cloning of similar fragments from different DNA donors.

Reference: Frengen, E. et al. (1999) A Modular, Positive Selection Bacterial Artificial Chromosome Vector with Multiple Cloning Sites. Genomics 58: 250-253. Reprints availiable upon request.

pBACe3.6 clones have chloramphenicol antibiotic resistance. Clones should be grown in LB containing 12.5 ug chloramphenicol/ml.

Research has shown that there is estimated to be an inherent ~15% error rate in the various genome databases which are used for the BAC clones. There also has been an estimated 2-3% cross contamination rate in the genomic libraries. When ordering a glycerol stock or agar stab clone from Empire Genomics, we select the requested clone from our freezers, and then we grow it overnight to confirm the clone viability and test it for T1-phage. Currently, we do not validate these clones for purity ( e.g. using methods such as PCR, fingerprinting or sequencing). In addition, we do not check public databases to find out which sequences have been reported for a particular clone, and do not determine if the sequences in the clone are consistent with the database. All DNA clone requests, however are streaked to single colony in our facility before growth and DNA preparation.

It is highly recommended that upon receipt of glycerol stocks and agar stabs, that you streak it to single colonies on a petri dish with LB agar and the appropriate antibiotic. Then use single colonies for DNA preparation, validation and replicate clones. In nearly all cases when a clone fails to meet expectations it is the result of an incorrect database entry or a cross contaminated clone. If it appears that there was a clone selection error on our part, we will replace the same clone and ship it to you free of charge.

Domestic (USA):$50.
International:$75+ depending on country.

PLEASE NOTE:

1) For large orders (especially dry ice), additional shipping cost may be applied.

2) EG cannot share the financial responsibility for delays or losses at the borders, or at any point during mailing.

Yes. We are required to include some Customs documentation with your clone shipment. To prepare this document, we require the following information from you: your name, mailing address with postal code, e-mail address, phone number, name of courier we are using (ie. FedEx), the number of packages being sent and the approximate weight/size; and a description of what we are sending (non-hazardous, non-toxic, non-infectious DNA only; the library name and the number of clones we are sending with approximate volume of each). Some countries do not accept dry ice shipments, please check with your distributor before placing an order. DNA is shipped with refrigerated packs, agar stabs in envelops at ambient temperature and glycerol stocks on dry ice. If you are shipping within the US, no such documentation is required.

Please visit ourBAC clone ordering pageto place all orders for RPCI BAC clones online. You can use our newCloneCentralsystem to quickly search for and locate BAC clones which cover a particular gene or chromosomal region of interest.

We have identified bacteriophage contamination in a small proportion of the BAC clone resource, apparently coliphage T1-related in most if not all cases. Phage T1 contamination can spread rapidly to other E.coli cultures causing lysis of the cells. We have put procedures in place to minimize the chance of transfer of phage contamination to users. We believe that as users of BAC and PAC clones you should be aware of the possibility of phage contamination and apply appropriate microbiological practices to ensure that your labs remain contamination free. We have withdrawn all contaminated sections of the resource from distribution. All clones requested will be tested for phage contamination before shipment, and only clones which test negative for phage will be distributed.

PLEASE NOTE:

1) All clones requested for distribution will be tested for Phage contamination, using a Top Agarose assay.

2) If the clones are non-lytic with the Top Agar assay, the clone(s) will be dispatched directly to the user.Even clones which have tested negative for phage should still be handled with care, as no phage assay can be guaranteed to be 100% accurate.

The BAC and PAC clones are processed and tested for T1 phage before distribution. The average turnaround time is 7-10 days. Occasionally this schedule will be delayed due to Holidays or the volume of orders we receive.

We prepare BAC and PAC DNA by utilizing an Autogen 740 plasmid purification system from 2ml O/N cultures. This method is a modified phenol:chloroform extraction and results in plasmid DNA of high quality. The DNA is resuspended in 100ul TE, and contains, on average, 5-10ug DNA. It is possible that some of the BAC DNA will linearize upon freeze/thawing; therefore attempts should be made to minimize this step. The DNA will not been treated with RNAse.

nformation about the clones you order can be obtained from the following genome mapping resources:

National Center for Biotechnology Information
UCSC Human Genome browser
Ensembl

All BAC and PAC clones are maintained in DH10B E.coli hosts. Clones derived from libraries constructed in the PAC vectors (pCYPAC2 or pPAC4) grow on medium with 25ug/ml Kanamycin. These libraries are RPCI 6, 21, 31, 91, and 92. Clones derived from all other RPCI libraries are constructed in the BAC vector pBACe3.6 and grow on medium with 20ug/ml chloramphenicol.

Single clones are distributed as DNA preparations, and/or agar stabs. Upon receipt, DNA should be stored at 4C,and agar stabs should be recultured as soon as possible after receipt.

The BAC and PAC libraries available at Empire Genomics are listedhere. We have every clone available from these libraries.All clones have identifiers that typically found by the end user as listed in various public databases and publications. The clone names must follow theNCBI clone nomenclature rules.

BAC and PAC clones from the RPCI human libraries were instrumental in deriving the sequence for the human genome, and have also proven to be optimal tools for fluorescence in situ hybridization (FISH)based studies (McPherson et al., 2001). Gross abnormalities can be mapped to the human sequence, thus delineating the specific sequence involved in a given genetic defect. The BAC Resource was developed through the National Cancer Institute (NCI) Cancer Chromosome Aberration project to enable cytogeneticists to finely map genomic aberrations by FISH-based analysis, and for application as array targets for high-resolution CGH. Additionally, the RPCI mouse libraries were utilized for the mouse genome project allowing a valuable resource for transgenic and knock-out mouse models. The large chromosomal segments carried in the BACs ensure that reproducible expression is retained for most genes after integration into the mouse genome. Other applications include PCR amplification, probe generation, fingerprinting and as template for end sequencing.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) cloning systems, developed in the 1990s, are ideal cloning systems for carrying DNA fragments 100 to 300 kb in size. These libraries, including the RPCI-11 human female library and RPCI-23 mouse male library, are amenable to genomic analysis, since their DNAs are easily prepared and manipulated (Osoegawa et al., 2001).

Yes. If no clones are located using your probe, we do not charge for the screening, only the probe generation.

In general, 2-3 weeks from receipt of probe to shipping of clones. We begin a hybridization experiment the first Monday of each month.

Yes, we can use cDNAs, PCR products, cloned genomic DNA and end probes from BACs. We need about 100 ng (25ng/ul) of purified probe for labeling. The probe should be free of contaminating vector, PCR primers, and other nucleic acids that may create non-specific signals.

The overgos designed are free of all known repetitive elements, E. Coli sequences, and vector sequences that would produce false-positive and non-specific hybridization-positive clones. The similar characteristics of each overgo designed also allows pooling of up to 50 overgos in a single hybridization experiment.

An efficient methodology for isolation of BAC clones was developed by J. McPherson at Washington University and modified by Empire Genomics. The approach involves hybridization to BAC library filters with short, radiolabeled probes. The probes are created from a pair of overlapping oligos and have hence been dubbed "overgos." Briefly, two 24mer oligonucleotides, derived from genomic sequence, which share eight base pairs of complementary sequence at their 3' ends are synthesized and annealed, creating a sixteen base overhang. This overhang is "filled in" using Klenow fragment thereby incorporating appropriate radionucleotides. The resulting product is a double-stranded 40mer with, on average, eight radiolabeled nucleotides incorporated per single-stranded product.

We can isolate a single clone(s) from a library address and provide BAC agar stabs and/or DNA preps. Up to three clones are included for distribution as part of the BAC screening service. Additional clones are available for purchase.

Using a custom-designed overgo generated from a sequence provided by you or a specific nucleic acid probe, we will screen the RPCI BAC library you specify and locate clones homologous to your probe. We usually locate 3-8 clones per unique probe.

An easy and safe way to ship total RNA samples is in RNAstable (from Biomatrica, http://www.biomatrica.com/rnastable.php). The product has been tested and found to be excellent for storage and shipping of RNA at room temperature when manufacturer instructions are carefully followed. After RNA is dried down in RNAstable, the sample must be enclosed in an airtight container or pouch with desiccant added to protect the RNA from ambient humidity. Your sample can be shipped at room temperature and for a much lower cost than shipping on dry ice. If the RNA does not have a preservative, please ship on dry ice and pack with adequate dry ice to keep RNA frozen for several days. International shipping routinely takes a minimum of two day and any delays in shipping will result in possible loss of sample integrity if an adequate amount of dry ice is not used. All shipments on dry ice should be mailed on a Monday or Tuesday to avoid having delayed shipments arrive on the weekend. The Empire Genomics facility does not receive samples over the weekend or during holidays

Who can I contact if I have questions?

You can contactinfo@empiregenomics.comwith your questions. Alternatively, we welcome your phone call at 716-845-3361 or toll free inthe US & Canadaat 1-800-715-5880.

Agilents websites (www.chem.agilent.com) have extensive technical support sections including FAQs on all steps of the protocols used, arrays available and a literature library section with papers, technical notes, and posters.

GeneSpring GX is a powerful visualization and analysis solution designed for use with gene expression data. The software provides advanced statistical tools, a range of visualization displays (scatterplots, pathway diagrams, and more), and data quality evaluation. Visit Agilents website for more information aboutGeneSpring GX.

Yes. Some act as negative controls and there is also a set of positive controls that are spiked into the hybridization cocktail.

They are 60 nucleotides long.

All of our technicians are experienced, highly trained individuals. Whenever possible, the same technician will perform all experiments in a project. Whenever possible we will use reagents from the same lot, arrays from the same print batch, and use the same equipment (hyb oven, scanners, etc) to reduce technical variability.

A typical experiment of 6-12 arrays takes about 1 week (including the basic data analysis service) from the time we receive the RNA and verify its integrity on the Agilent Bioanalyzer.

The decision to use 1-color or 2-color is mainly a factor of the experimental design and the research question asked. 1-color experiments are generally less expensive as less labeling reagents are required. In addition, a control RNA is not required on each array, often preserving precious sample. 2-color experiments generate results in a ratio format rather than absolute numbers. Technical variation between arrays is automatically accounted for since a reference RNA is used for each co-hybridization.

Yes, all fluorescently labeled probes are quality control checked on a Nanodrop ND-1000 spectrophotometer for appropriate incorporation before proceeding with hybridization.

All RNA samples should have a Bioanalyzer 2100 rRNA [28S/18S] ratio > 1.5, RIN >7 to pass QC. Occasionally, researchers will carry on with an experiment even if the RNA is slightly degraded knowing that this is the highest quality RNA they can isolate. Empire Genomics will notify you prior to starting the gene expression process if the RNA fails QC.

Assessing the quality of your RNA sample before proceeding with labeling and hybridization can save a lot of time and money. It can also reduce the time spent troubleshooting if something goes wrong at the next step. The Agilent Bioanalyzer is a convenient replacement for standard denaturing agarose gels; it is much faster and requires only 1?L at a concentration of ~100 ng/?L for analysis. The Bioanalyzer uses capillary electrophoresis (lab-on-a-chip technology) to move the sample through a gel matrix. The resultant electropherogram provides the user with a detailed trace of each sample and a metric of RNA quality (RIN).

We can amplifiy 50-100 ng of total RNA using the Low RNA Input Linear Amplification Kit from Agilent Technologies. This method requires only one round of amplification and produces either cyanine-labeled cRNA (anti-sense) or cDNA (sense) from total RNA, depending on RNA source.

We require a minimum of 2?g of total RNA (50-250ng/ul, treated with DNase I, 260/280: 1.9-2.1) per labeling reaction. This amount will also allow us to perform quality control analysis using the Agilent Bioanalyzer

A technical replicate involves the multiple labeling or reciprocal labeling of thesameRNA sample. The purpose of a technical replicate is to control for technical variability within an experiment (array to array variation, reagent variation, dye incorporation, etc.) A well run experiment should have minimal variation thus minimizing the need for technical replicates.A biological replicate involves isolating RNA independently from replicate sources (multiple cell lines, multiple biopsies, multiple patients, etc). The purpose of a biological replicate is to control for biological diversity. Biological replicates are often more telling, and for this reason are better than technical replicates, however, biological replicates are often more difficult to obtain.

Dye-flip (reciprocal) labeling is recommended for expression analysis when fluorescently labeling cDNA as the dyes incorporate with different efficiencies.

We recommend that you consult with a biostatistician to determine the proper number of replicates. Typically, three replicate experiments are required for small sample sizes if data analysis is to be performed with even minimal confidence. Larger sample sizes may only require a single replicate experiment.

Yes, you can contactinfo@empiregenomics.comwith your specific questions. You can also call us at 716-845-3361 or toll free in Canada/U.S.A. at 1-800-715-5880.

Yes. If you are shipping RNA/DNA to US from outside of the US you are required to include some Customs documentation with your shipment. To prepare this document, we require the following information from you: your name, mailing address with postal code, e-mail address, phone number, name of courier you are using (ie. FedEx), the number of packages being sent and the approximate weight/size; and a description of what you are sending (non-hazardous, non-toxic, non-infectious RNA/DNA only; please specify the organism the RNA/DNA is from and the number of tubes you are sending with approximate volume of each). Please indicate the samples as having zero value.If you are shipping within the US, no such documentation is required.

Please send your RNA samples, along with the appropriate paperwork, to:Empire Genomics, LLC Attn: Jeff Conroy Microarray and Genomics Facility BLSC Rm L1-313 Elm & Carlton Sts Buffalo, NY 14263 USA

Place your sample in a Styrofoam cooler with cold packs or dry ice. DO NOT USE WET ICE TO REFRIGERATE YOUR SAMPLES. Place enough cushioning material around your sample to prevent movement within the Styrofoam cooler. This will further reduce the possibility of your sample being damaged by the ice pack, or dry ice, bouncing around and crushing your sample tubes. Place your Styrofoam cooler inside of a cardboard box to ensure acceptance by carrier. Packages should be shipped overnight Monday-Thursday to ensure that samples are received by Friday. The Empire Genomics facility does not receive samples over the weekend or during holidays.

A good sample will have a 260/280 ratio of 1.8-2.0, and a 260/230 ratio of >2.

The required amount of DNA is 1.5 g in a concentration of 100-300ng/ul.

Yes. If you are shipping RNA/DNA to US from outside of the US you are required to include some Customs documentation with your shipment. To prepare this document, we require the following information from you: your name, mailing address with postal code, e-mail address, phone number, name of courier you are using (ie. FedEx), the number of packages being sent and the approximate weight/size; and a description of what you are sending (non-hazardous, non-toxic, non-infectious RNA/DNA only; please specify the organism the RNA/DNA is from and the number of tubes you are sending with approximate volume of each). Please indicate the samples as having zero value.

If you are shipping within the US, no such documentation is required.

Please send your DNA samples, along with the appropriate paperwork, to:

Empire Genomics, LLC
Attn: Jeff Conroy
Microarray and Genomics Facility
BLSC Rm L1-313
Elm & Carlton Sts
Buffalo, NY 14263 USA

We will email you a username and password to access our secure results center so you can download your aCGH data.

Agilent selects a small number of service labs to participate in a certification process involving on-site training as well as assessing technical ability throughout the protocols. Empire Genomics successfully completed this training in August 2006.

Yes, aCGH services are available using our Human ACCUArray, and Mouse ACCUArray,and Agilent platforms. Typical turnaround times are 3-4 business days.

Yes, we manufacture and offer a 19K Human BAC array and 6K Mouse BAC array.

Fluorescent in situ hybridisations (FISH) experiments are done to confirm deletions and duplications identified by microarray analysis.

Wecan perform whole genome amplification (WGA) of low-yield DNA samples to produce the required 1ug from as little as 50ng template DNA. The DNA can be from either fresh, frozen or FFPE samples.

Werequire at least 1ug of high molecular weight DNA for both the Agilent and BAC arrays. Most standard genomic DNA isolation protocols will yield DNA of high enough purity for aCGH. It is highly recommended to check genomic DNA for degradation and RNA contamination. Low molecular weight DNA can be used (paraffin embedded, formalin fixed, degraded, etc.) but results may be more unreliable. All DNA samples are checked by for quality prior to aCGH analysis.

There are two main types of spotted aCGH arrays: BAC (150 to 200kb) and oligonucleotide (25-85 bp).

BAC Strengths:BAC arrays offer many advantages for aCGH analysis. Each BAC clone represents ~171kb of genomic sequence that serves as a reliable hybridization target. The BAC clone targets have been mapped to the human reference sequence produced by the International Human Genome Sequencing Consortium, allowing easy access to information in the related genomic databases. The LM-PCR products that are generated to represent the BACs are of a size and complexity that increases hybridization signal and therefore accuracy in identifying true copy number changes. (No need for replica experiments). Using BAC aCGH, genome wide scans of sample DNA can be accomplished within 24 hours even with difficult samples that are often heterogeneous, harbor varying degrees of aneuploidy, or are derived from FFPE archive material (see figure T). Additionally, the BACs that are identified can also be used directly as FISH probes for hybridization to nuclei or metaphase chromosomes for copy number validation. BAC aCGH has the power to detect CNAs in samples with 50% contaminating normal cells (see Figure X). Segmental amplification of whole chromosome arms, terminal deletions, and discrete, high magnitude copy number gains and losses are easily detected (Figure Y), making the interpretation of the breakpoints very straight forward.

For diagnostic applications, standardization, reproducibility and validation are important issues. BAC arrays have the advantage of the most reproducible platform as seen in the MARG study. This reproducibility has led to the standardization of protocols for probe labeling, hybridization and data analysis. Each clone on the array can be further utilized as a FISH probe for CNA validation, and is available through EG for distribution.

BAC Limitations:BAC arrays are unable to distinguish deletions/amplification less than ~85 kb or 50% of a BAC. As with all CGH platforms, the arrays cannot detect epigenetic changes, balanced translocations, mitochondrial DNA aberrations or point mutations. It also does not define the origin of translocation events or complex chromosomal rearrangements.

Oligonucleotide Strengths:The Agilent oligonucleotide arrays offer genome-covering resolution that can offer precise delineation of breakpoints. This is important in determining common regions of overlap and genes implicated in CNAs. These arrays contain 60mer targets that are synthesized on the slides at densities of 244,000 features per chip. EG, as an Agilent Certified Microarray Service Provider, offers full service human and mouse 44K and 244K oligo aCGH analysis.

Oligonucleotide Limitations:Due to their small target size, oligonucleotide arrays suffer from poorer signal to noise ratios that often results in a significant number of false-positive outliers (see BAC, Agilent and Affymetrix data in section 2). Typically 46 adjacent oligonucleotides are necessary for a reliable call thus identification of regions of CNA requires the use of moving average algorithms. This computational smoothing of oligonucleotides reduces the spatial resolution and can be difficult to standardize over large data sets. Repeat hybridizations are often recommended to resolve false-positive outliers. Small target sizes also minimize effectiveness with degraded DNA or DNA prepared from FFPE tissue and makes them more susceptible to cross-hybridization with multiple genomic loci.

A tiling array is one that covers most of the genome using overlapping BACs. The very high resolution of this type of array allows gains or losses of 40-80 kb regions to be detected.

If an aCGH array has a 100kb resolution, it means the array has approximately one element (BAC or oligonucleotide) spotted on the slide per 100 kilobase of the genome.

Array Comparative Genomic Hybridization (aCGH) provides rapid (24-48 hour) analysis at high resolution on sample DNA. The information provided by aCGH analysis can be directly linked to physical and genetic maps of the human and mouse genomes for the precise mapping of breakpoints and gene content. Traditional cytogenetics and CGH has limited resolution, 7-10 day turnaround times and typically requires dividing cells.

Array Comparative Genomic Hybridization (aCGH)provides a high resolution, highly efficient means to detect, quantitate and map regions of abnormal copy number (both gains and losses) by comparing the relative efficiency with which fluorescently-labeled test and reference DNA hybridize across the genome. aCGH allows for the analyses of DNA copy number in a variety of sources including, frozen trimmed human tumor cell blocks, microdissected frozen and Formalin-Fixed Paraffin-Embedded (FFPE) Tissue, cell lines, lymphocytes, and flow sorted cells. aCGH has been used successfully for the submicroscopic analysis of autism, mental health disorders, cancer and constitutional chromosome abnormalities (microdeletion syndromes). Genome-wide arrays cover the entire genome allowing for identification of chromosomal changes in suspected and novel regions.

Array comparative genomic hybridization (aCGH) is a microarray-based technique that utilizes discrete segments of DNA as immobilized targets to identify genome-wide chromosomal copy number changes (amplifications and deletions). These DNA targets include oligonucleotides (i.e. Agilent, NimbleGen and Affymetrix platforms), PCR amplified products from cDNA clones or complex PCR representations of Bacterial Artificial Chromosomes (BACs).

BAC and PAC clones from the RPCI human libraries were instrumental in deriving the sequence for the human genome, and have also proven to be optimal tools for fluorescence in situ hybridization (FISH)-based studies (McPherson et al., 2001). Gross abnormalities can be mapped to the human sequence, thus delineating the specific sequence involved in a given genetic defect. The BAC Resource was developed through the National Cancer Institute (NCI) Cancer Chromosome Aberration project to enable cytogeneticists to finely map genomic aberrations by FISH-based analysis, and for application as array targets for high-resolution CGH. Additionally, the RPCI mouse libraries were utilized for the mouse genome project allowing a valuable resource for transgenic and knock-out mouse models. The large chromosomal segments carried in the BACs ensure that reproducible expression is retained for most genes after integration into the mouse genome. Other applications include PCR amplification, probe generation, fingerprinting and as template for end sequencing.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) cloning systems, developed in the 1990s, are ideal cloning systems for carrying DNA fragments 100 to 300 kb in size. These libraries, including the RPCI-11 human female library and RPCI-23 mouse male library, are amenable to genomic analysis, since their DNAs are easily prepared and manipulated (Osoegawa et al., 2001).