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Frequently Asked Questions

Hybridization/Probe Questions:

Slide Questions:

Ordering Questions:

Software/Analysis Questions:

RNA/Control Transcripts Questions:


Hybridization/Probe Questions:

Is it okay to use probes with calculated dye incorporations lower than what you suggest from your protocol?

For genomic hybridizations, the PFGRC uses dye incorporation in excess of 300 pmoles. For RNA probes dye incorporation should be at least 800 to 1000 pmoles. The more incorporated dye put on a slide, the better the hybridization results.

Others I have talked to hybridize at 65 degree in the absence of formamide. Have you found better results using formamide at 42 degrees?

No. We have experienced difficulties with probes drying out under the coverslip at high hybridization temperatures.

The TIGR protocols for hybridization (date 9/02) were, I believe, developed using PCR product arrays. Have these protocols been tested and found to work well on oligonucleotide arrays?

These protocols have successfully been used on both amplicon and oligonucleotide microarrays.

Have you encountered any problems using LifterSlips™?

The main concern with LifterSlips™ is determining which side the has the ridge. Proper placement is important as all cover slips can scratch the surface of the slide. When adding probe to slides with LifterSlips™ it may be easier to add the probe next to slip and allow it to be drawn under the slip by capillary action.

Can I use reagents/materials from a different manufacturer than what is listed on your protocol? Can I use a different concentration than listed on the protocol?

The SOPs that the PFGRC have developed have worked well for the PFGRC for years and are constantly being evaluated and improved. If an investigator wishes to alter concentrations or materials they can do so understanding that changes in concentrations may require further optimization of probe generation or hybridization protocols.

What type and size of cover slip do you recommend for performing hybridizations?

Currently the PFGRC uses Erie Scientific LifterSlips™ 25mM X 60mM. Fisher Scientific glass cover slips can also be used if LifterSlips™ are not desired.

Should I optimize my aminoallyl-dUTP to dTTP ratio for my organism?

The 2:3 and 2:1 aminoallyl-dUTP to dTTP ratios work well for a wide range of organisms, however certain organisms may require detailed optimization.

Slide Questions:

What kind of slides are you using to print your arrays?

Currently, the PFGRC is using amino-silane Nexterion A Schott slides. Since slides are constantly being evaluated, vendors and slides may be altered if slides do not perform to PFGRC standards or if improved products become available.

Do the arrays need any further processing (e.g. UV cross-linking) prior to use?

PFGRC slides can be used immediately. Immobilization of the DNA has occurred prior to shipping. Investigators should begin slide processing at the pre-hybridization.

What is the coating the slides are prepared with?

Currently the PFGRC has both amino-siliane and epoxy based slides in inventory.

What is the shelf life of the slides?

Once printed, up to a year. Slides which are a year old may still be viable.

What is the post-printing processing (rehydration, baking, crosslinking) that you do to your slides?

Once printed slides are allowed to dry slowly at 65% humidity to insure good spot morphology. Slides are UV cross-linked at 25000uJ (25mJ) and stored in a desiccator until shipped.

Could you please tell me how many replicate spots will be on the slide for a gene?

Spot replicates are dependant on the organism size. Currently the PFGRC printing capacity is 10,000 spots per slide. Small organisms may be printed 4X while larger organism may be printed 2X or even 1X.

Is my microarray a 70-mer oligonucleotide microarray or the amplicon based microarray?

Version 1 and 2 arrays for Salmonella typhimurium/typhi, Staphylococcus aureus and Streptococcus pneumoniae are amplicon arrays. All other organisms and versions are 70mer-based oligonucleotide arrays.

What are the sizes of the PCR products that are printed on the amplicon-based micrarrays?

Amplicon sizes can range between 50 to a 1000 base pairs.

What is the "test" array?

Test arrays are provided so that the user can become familiar with the practice of hybridization, probe creation and data analysis. Test arrays will be provided as required by the user. There are 24 grids per slide with each column representing a single ORF repeated 15 times. Test arrays consist of a total of 380 open reading frames taken from each of three reference organisms:

  • Staphylococcus aureus reference strain COL
  • Streptococcus pneumoniae reference strain TIGR4
  • Salmonella typhimurium reference strain LT2
A test and control set of RNA from Streptococcus pneumoniae will be provided to the user as practice in generating indirect labeled cDNA probes for use on a microbial array.

Which side is the printing located on and where are the location of the grids?

Slides are printed on the bar-coded side of the slide. The bar-code is considered the bottom of the slide. The printing area is approximately 4mM in from both sides and the top of the slide.

How should the slides be stored upon arrival?

Slides arrive in a foil seal with desiccant. Please store sealed slides in desiccator until use. Once opened store unused slides in desiccator. For optimal results slide should be used within 6 months to 1 year.

What quality control methods do you use to assure the quality of your arrays?

Multiple slides from each printing of 75 slides are quality controlled using IDT SpotQC. Images are reviewed for printing irregularities, spot morphology and DNA retention. If necessary, hybridizations can be performed to test array performance. Quality control images are available upon request.

Ordering Questions:

What is the timeframe from ordering to receiving my arrays?

Usually shipping turn around is 2 days with orders shipped two day FedEx. Orders are not shipped on Friday due to shipping limitations.

Is there a limit on the number of test arrays I can order?

Since test arrays are for optimization and learning purposes, they are unlimited.

Where do I place my order for slides I have been approved for?

Slides can be ordered off of the PFGRC's web-site under "investigator login". Both the investigator's login and password are required.

Do I need to have my compliance form completed before I can place an order for microarrays?

Compliance forms must be submitted and approved before slides can be ordered.

Can I order all my slides at once?

It is strongly suggested to order organism slides in sets of 25 or 50. By ordering in small quantities, we can insure that we provide the freshest possible slides.

Software/Analysis Questions:

Where can I find information about grid parameters for TIGR Spotfinder?

Information about grid parameters for all PFGRC microarrays are accessible from the Available Microarrays page. Select the organism and microarray version you are interested in and scroll down to the table with the Spotfinder grid parameters.

Are there different printing patterns available for my array?

Each organism version has a constant specific printing pattern.

Where can I find microarray annotation files?

Annotation files for all PFGRC microarrays are accessible from the Available Microarrays page. Select the organism and microarray version you are interested in and scroll down to see the links to the annotation files.

What are these "black holes" in the array?

"Black holes" or negative spots on a slide indicate poor pre-hybridization blocking, low dye incorporation or impure starting material. Negative spots are caused when the spotted DNA is blocked and the probe is binding to the background surface.

Why can I not see any spots after scanning.

Ensure slide is being scanned at an adequate PMT. The main reasons for no signal is low dye incorporation or impure starting material which is inhibiting the hybridization reaction. Ensure that the starting material for probe generation is clean and not degraded. Starting with clean RNA and DNA is essential to produce a good hybridization.

I am able to see background outside the spots, but why are all of my spots completely black?

Same answer as "Black holes" question.

What is the reasonable level of background I can expect on my array?

Reasonable background level is one that doesn't overpower the spots on the array. Good background level is around 1000 with some saturated spots. Excellent background level is 200 with a few saturated spots. To achieve reliable data signal to background ratios should be 2 fold.

Am I required to use your microarray analysis software? Are there other microarray analysis software programs available that you recommend which I can download for free?

No, no one is required to use the software provided by TIGR or recommended by us. Investigators should feel free to use their own software packages for data analysis. However, it is much easier for the PFGRC to understand analysis issues on software packages that are familiar to us. There are a number of free programs for doing analysis produced by TIGR at http://www.jcvi.org/cms/research/software/ . PFGRC recommends the use of Spotfinder http://www.jcvi.org/cms/research/software/ and MEV http://www.tm4.org/mev/ for doing microarray data analysis.

Im a using a different software analysis tool. Do you have an annotation file that is compatible with my software? What file formats do you support?

The PFGRC currently supports .GAL files (Gene Pix Array List) or tab-delimited files which can easily be imported to any spreadsheet program.

What do you consider to be a unique gene?

Unique ORFs are defined as one of the following:

  • Truly unique sequences present in one strain but none of the other strains.
  • Differences representing polymorphisms in gene length.
  • Annotation differences between strains, due to method of annotation.
  • Sequence divergence between similar genes shared between multiple strains.
Our philosophy has been to be inclusive of as many ORFs as possible in the design of the microarray.

What coverage of the genome do your chips cover?

We attempt to design a unique reporter oligo for every ORF in the annotated genome sequence. For some ORFs this may not be possible. Therefore we attempt to get the widest coverage possible of the genome's annotated compliment while retaining specificity of sequence with the 70-mers that we design. For chips where there is more than one strain we design 70-mers that represent a unique hit across all the strains on the chip. Where there are strain-specific ORFs we have also attempted to design 70-mers to hit those ORFs as well.

I need some training on the TM4 microarray software suite. Does the PFGRC offer training classes at the JCVI?

Yes, we offer several training courses that include TM4 content. Please visit the Education page for information about the course offerings and their content.

What type of scanners do you use and have you had success with these?

The PFGRC uses Axon 4000B scanners.

How can I interpret the results from the test array?

Upon request the PFGRC will provide a analysis of a microarray hybridization and data analysis of the test array and the test RNA (done with TIGR Spotfinder). This is so that investigators can compare results from hybridizations on the test array with results obtained from their own work with this array.

RNA/Control Transcripts Questions:

How do I use the control transcripts RNA?

To use the control transcripts, simply add 10 ng of the control transcript(s) to the RNA labeling reaction . Multiple control transcripts can be spiked in, as long as the volume of RNA, random hexamers and transcripts does not exceed 18.5 Ál as indicated in step 5.1.1 of SOP M007, Microbial RNA Aminoallyl Labeling for Microarrays. Other than this initial spiking, no further steps need to be taken to generate the transcript probe. Simply perform SOP M007 and hybridize the labeled probe using SOP M008. When analyzing, use the Test Array annotation file to determine if the pattern was generated correctly.

What controls do I have on my microarray?

The ten control spots are only on the test arrays. All other arrays have the 500 A. thaliana genes as controls.

What amount of control transcripts would be appropriate to add to our experimental RNA?

The PFGRC recommends using 10ng per control transcript used.

The test RNA sample arrived shipped at room temperature, is this correct?

Yes. RNA for the test arrays is shipped in salt and ethanol, thus a precipitation step must be performed prior to use.

Have you observed any cross-hybridization between the control transcripts/universal references with DNA from the array organisms?

It takes as little as 15 bases of similar sequence to generate cross-hybridization. Nonetheless so far, we have experienced little to no cross-hybridization between organism DNA and the universal references.

How do you harvest RNA?

The PFGRC uses Hot-Phenol Acid method to extract RNA.

What are the reserved spots on your arrays?

The reserved spots are 70mers of 500 A. thaliana genes. A control mix for these controls is currently in R&D.

Is my RNA or DNA purity important for my probe production?

This is far and away the most critical aspect of microarrays. 95% of all microarray problems can be traced back to the starting material. DNA and RNA purity is absolutely critical. Any residual chemicals from isolation, residual proteins, RNA/DNA contamination or degradation can affect probe generation and hybridization efficiency. Pure material will reproducibly generate high signal and low background. Less than ideal material will produce probes of varying quality and background issues. Unfortunately 260/280 ratios, gels and Bioanalyzer images are not always adequate indicators of material quality. If there is any doubt concerning probe quality the starting material should be cleaned, sometimes several times. If cleaning does not help, sample may need to be reisolated.

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