3DNA Array Detection
Frequently Asked Questions
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Which kit should I use for my microarray experiments? |
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Consider the amount and type of your RNA sample, your method of hybridization (manual or hybridization station) and the desired method of priming when choosing a kit. See our Kit Selection Guide. |
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What is the difference between dT priming and random priming? |
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dT priming is appropriate for messages that contain a 3’ poly(A) tail. Since the dT primer starts synthesizing cDNA from the 3’ end, the cDNA may demonstrate a 3’ bias. If your array is printed with cDNA synthesized using 3’ primers, you may want to use dT priming for your samples. Random priming is appropriate for aRNA, partially degraded RNA samples, and messages that do not contain a 3’ poly(A) tail. The random primer will prime across the entire length of the message, and will generate several short fragments of cDNA from the message. |
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What is the shelf-life of my 3DNA kit? |
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Every 3DNA kit contains an expiration date on the side of the package. The expiration date will be 3 - 6 months after purchase if stored as directed in the protocol. If stored as directed, some of the components may be used up to 1 year after purchase. These components include the RT primer, hybridization buffers, LNA dT blocker, and capture reagents. However, the dNTP mix, Superase-In RNAse inhibitor, dTTP, TdT enzyme and T4 DNA Ligase should not be used after the expiration date. Please consult with Technical Support prior to using an expired kit. |
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Which scanners are compatible with your products? |
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Based on customer feedback, all commercially available scanners are compatible with our products. The intensity of the signal will vary from scanner to scanner because of differences between the hardware (lasers, filters, etc.) used by each manufacturer. Consult the User’s Guide for your scanner for recommended laser and PMT settings prior to scanning. |
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What array surfaces have been successfully tested with Genisphere kits? |
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Epoxy (MWG™, GeneMachine™)
Aminosilane (Telechem™, Corning GAPSII™, Corning Ultra GAPS™)
Aldehyde
Poly-L-lysine
Corning GLYMO Epoxy™ |
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What species of RNA have been successfully tested with Genisphere kits? |
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| Human |
Maize |
E. coli |
| Rat |
Cotton |
Staphylococus Aureus |
| Mouse |
Spruce |
Canine |
| Drosophila |
Tomato |
Potato |
| Arabidopsis |
Yeast |
Tobacco |
Note: Some species of RNA will perform better with a particular reverse transcriptase. Please contact Technical Support when choosing a reverse transcriptase. In addition, the preparation method of the RNA sample may also contribute to its compatibility with Genisphere labeling kits. See the following reference:
Ruuska, S.A. and Ohlrogge, J.B., Protocol for Small-Scale RNA Isolation and Transcription Profiling of Developing Arabidopsis Seeds. BioTechniques, 31(4):752-758, 2001.
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Which hybridization buffer should I use? |
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We provide an SDS-based hybridization buffer and a Formamide-based hybridization buffer with each kit. As long as the buffers are used within the appropriate temperature ranges (see protocol) there should be no difference between the results they yield. On some mirror coated slides or poly-L-lysine coated slides, the coating may begin to peel off at the hybridization temperature required for use with the SDS-based buffer. If you experience this problem, use the Formamide-based buffer as directed. The protocol contains recommendations for use of both buffers. Please contact Technical Support to check for compatibility of in-house or other commercial buffers with Genisphere kits. Some Genisphere kits also contain Enhanced Hybridization Buffer, which can be used in the cDNA hybridization when additional sensitivity is desired. |
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Do I need to use competitor DNA (C0T1, sheared salmon sperm, etc.) in my hybridizations? |
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Competitor DNA is used to block repetitive sequences that may be found on an array. The use of competitor DNA is optional. You may determine on your own if competitor DNA should be a part of your assay. |
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Do I need to use the LNA dT blocker in my hybridizations? |
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The LNA dT blocker is required for all cDNA arrays. It may not be necessary to use the LNA dT blocker for oligo arrays, but it will not harm your assay if it is used. |
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Which reverse transcriptase should I use? |
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If you are currently running microarrays and are using a particular reverse transcriptase successfully, we recommend that you continue to use that enzyme with our kits. Alternatively, we recommend the use of SuperScript II reverse transcriptase (Invitrogen Cat. No. 18064-014: 10,000 Units @ 200 U/mL). You can also use a commercially available reverse transcriptase from Genisphere (Cat. No. RT300320), Promega, Clontech, or Stratagene. |
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Is there any way to check if my cDNA was successfully made, before putting my samples down on my arrays? |
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We recommend checking the synthesis of cDNA by running the equivalent of 1 ug of total RNA on a 6-10% denaturing polyacrylamide gel and staining with Sybr Gold (Molecular Probes). The cDNA will appear as a smear between 500-5000 base pairs. Click here for an example |
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Can I use more capture reagents and receive a better signal? |
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We have done extensive work to determine the optimal amount of capture reagent that should be used to generate the best signal. All Genisphere protocols indicate the appropriate amount of RNA to use, and the appropriate hybridization volumes. As long as these parameters are followed, the flourescent signal will not improve when more than 2.5uL of the capture reagents are used. If you need to use a larger quantity of RNA, or a larger hybridization volume than what the protocol recommends, contact Technical Support. |
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What is the linear range of Genisphere microarray products? |
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Click here to see linear range data. |
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Will the 3DNA dendrimer bind to specific sequences on my array without cDNA? |
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Under the correct hybridization and wash conditions (see the protocols), you should not experience any non-specific binding of the dendrimer to your array. The dendrimer sequences are designed to be unique (abiotic). However, on some oligo arrays, you may observe some binding of the dendrimer to certain oligos. If this occurs, contact Technical Support for recommendations. |
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How can I prevent fading of Cy5/Alexa Flour 647/Oyster-650? |
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Cy5 dye performance may be affected by a variety of factors that are particularly prevalent during the summer months. Exposure of the Cy5 dye solutions and the hybridized arrays to light and to oxidative environments may cause rapid fading of the Cy5 dye, regardless of the labeling system used. Limiting or controlling the exposure of the arrays to these environments has been shown to significantly reduce Cy5 fading. Please consult the appendix of your protocol or contact Technical Support for details on the following recommendations:
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Always keep solutions and arrays containing Cy5 away from light, including normal fluorescent lighting. |
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Use the Anti-Fade Reagent (provided with the 3DNA kits) in the hybridization solution containing the Cy5 Capture Reagent. |
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Do not use plastic coverslips or a hybridization chamber in which plastic parts come in contact with your hybridization mixes. |
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Use a high quality, non-DEPC treated nuclease free water to make your solutions for post-hybridization washes. As noted in the Internet List Serve, MilliQ® water has been shown to damage Cy5 (http://groups.yahoo.com/group/microarray/ messages/2867). Water from Ambion and VWR (Cat. No. RC91505) have been validated for use with microarrays and do not contain components that will oxidize Cy5. |
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Add dithiothreotol (DTT) at a final concentration of 0.1mM to the post-hybridization wash solutions. |
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Be sure to follow the protocol for proper preparation (thawing, mixing, and heating) of all 3DNA Capture Reagents before each use. |
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Use DyeSaver2 (Genisphere Cat. No. Q500500) immediately after the final washing and drying of the microarray to preserve fluorescent signal. |
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Can I substitute the components of one 3DNA kit for another 3DNA kit? |
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We do not advise exchanging components, because some components are incompatible between kit types. If you have more than one type of kit, please contact Technical Support. |
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Which 3DNA kits are compatible with Hybridization Stations? |
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Array 900HS
Array 350HS
Array 350RP
Array 50 Version 2
Please contact Technical Support if you would like to confirm kit compatibility, or if you need a protocol for hybridization stations. |
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How should I isolate and purify my RNA before using a 3DNA kit? |
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Many different protocols, as well as commercially available kits from Qiagen, Ambion, and Life Technologies, have been used successfully with Genisphere kits. After purifying your RNA, we recommend that you check your RNA sample for degradation and genomic DNA contamination on an agarose gel. These methods are outlined in Genisphere protocols. |
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Should I treat my RNA sample with DNase? |
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While treating an RNA sample with DNase is not required for Genisphere labeling, it is a good practice since your RNA concentration will be more accurate. Prior to treating your RNA sample with DNase, you should check your sample with a spectrophotometer. The OD 260/280 ratio should be between 1.9 and 2.1. You should also run your RNA sample on a gel to check for genomic contamination and degradation of your sample. You should only treat your RNA sample with DNase if you have genomic DNA contamination. After treatment with DNAse, you must completely eliminate the DNase before making your cDNA, since DNase will destroy your cDNA product. Methods for inactivating DNase include phenol-chloroform extraction and RNeasy® kits from Qiagen, following the DNase treatment. High temperature treatment of DNase may not completely inactivate the enzyme. |
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Should I use Alexa Flour or Cy dyes? |
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The choice of which dyes to use is one that the end user will need to determine. In our experience, the dyes tend to perform similarly on microarrays. In some cases, one set of dyes may perfrom better than the other under a given set of conditions. Genisphere reccommends consulting with both Amersham™ and Molecular Probes™ to determine wich set of dyes would be most appropriate. |
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What is a 3DNA dendrimer? |
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A 3DNA dendrimer is a signal amplification molecule made from DNA. Each dendrimer has a “core” that consists of a matrix of double-stranded DNA, as well as an outer surface comprised of hundreds of single-stranded arms. The surface arms are available for hybridization to a specific sequence as well as oligonucleotides that carry signal molecules. For a more detailed description of 3DNA technology see Architecture of 3DNA Dendrimer. |
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How are fluorescent dyes attached to the dendrimer? |
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Fluorescent dyes are covalently attached by coupling fluorescently-labeled oligonucleotides to the free surface arms of each dendrimer. The excess labeled oligonucleotides are then removed by chromatography. Each lot of fluorescent dendrimer undergoes quality control testing using gel electrophoresis, fluorometry, and microarray performance prior to release. |
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How much “lot to lot” variation occurs in the number of dyes per dendrimer? |
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The variation is less than 5.0%. Here is a table taken from a peer-reviewed article which summarizes the preparation of five individual Cy3 “lots” and five individual Cy5 “lots” of dendrimer.
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With so many dyes attached to each 3DNA dendrimer, does quenching occur? |
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Quenching occurs when fluorescent dyes are close enough together that the energy that would be emitted as fluorescence from one dye is transferred to a neighboring dye molecule, instead of being emitted. The dyes on the arms of the dendrimer are separated by enough nucleotides that quenching is not observed in 3DNA dendrimers. We have verified this result by following the fluorescence of a fixed quantity of dye molecules before and after attachment to dendrimers. The fluorescence was maintained. |
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Do the 3DNA reagents experience photobleaching? |
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Many fluorescent dyes experience photobleaching, or fading, of the signal after repeated excitation-emission cycles. The fluorescent dye molecules attached to 3DNA reagents are chemically similar to their dNTP counterparts, and will fade over time. Ways to avoid photobleaching include protecting the dyes from light and avoiding excessive laser power settings on some scanners. For a further explanation please contact Technical Support. |
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How big is the 3DNA dendrimer? |
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The Array 900 and 350 series kits contain four-layer dendrimers. A four layer dendrimer theoretically has 324 single stranded DNA arms in the outer layer. The diameter of a four layer dendrimer is 182-190 nm and the molecular weight is 1.2 x 107 Daltons. The Array 50 and FISH products contain two layer dendrimers. A two layer dendrimer theoretically has 45 single stranded DNA arms in the outer layer. The diameter of a two layer dendrimer is 70-90 nm and the molecular weight is 1.3 x 106 Daltons. We can make dendrimers in a wide range of sizes, but have found that the above sizes work most appropriately for their associated applications. |
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What other applications use 3DNA dendrimer technology? |
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3DNA dendrimers have been used for:
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1. gene expression arrays |
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2. in situ hybridization |
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3. microRNA detection |
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4. nucleic acid blot hybridization (Southern, Northern, Slot, Dot blots, and Western) |
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5. Biosensor |
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6.CGH |
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7. PCR |
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8. ELISA |
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9. Flow Cytometry |
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10. Bead-based assays |
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11. Protein Arrays |
3DNA dendrimers may have potential to be used in other assay formats. Please contact Technical Support if you wish to try an application that has not been listed, so we can provide recommendations. |
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