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Submicro
Expression Array Detection Kits
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| Table 1. Dendrimers have a consistent number of fluorescent molecules | ||
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2 3 4 5 |
256 242 247 244 |
243 237 256 238 |
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SE |
4 |
4 |
| Quantitative analysis of 6 different lots of Cy3- and Cy5-labeled dendrimers. SE, standard error. | ||
With so many dyes attached to each 3DNA dendrimer, does quenching occur?
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. Since the dyes on the arms of the dendrimer are at least 30 nucleotides apart, quenching is not observed. 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.
Do you experience photobleaching with the 3DNA reagents?
All fluorescent dyes will 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, so there will be fading over time. For a further explanation please contact our technical support line.
Can 3DNA dendrimers be used on membrane arrays?
Yes, but the protocol will differ somewhat from our Submicro (glass-slide) microarray protocol. The membrane protocols are currently under development and you should call technical support for more information.
Will the 3DNA dendrimer without cDNA bind to specific sequences on my array?
Under the correct hybridization and wash conditions (see the protocol), you should not experience any non-specific binding of the dendrimer to your array. The dendrimer sequences are designed to be unique (abiotic).
What is the shelf-life of the Submicro kit?
Every 3DNA kit contains an expiration date on the side of the package. Generally, the expiration date will be 2-6 months after purchase if stored as directed in the protocol. If stored as directed, some of the components, like the RT primer (Vial 2), hybridization buffers (Vials 6 and 7), dT blocker (Vial 9), and possibly the fluorescent 3DNA reagent (Vial 1), may be used after the expiration date. However, the RT enzyme (Vial 3) and the dNTP's (Vial 4) should not be used after the expiration date. In any case you should consult with tech support prior to using an expired kit.
I looked at the fluorescently-labeled Vial 1 reagent and it does not have any color?
The fluorescent 3DNA reagent in Vial 1 does have color. However, it is much less intense than the color you are used to when using a dNTP dye. The dNTPs that are used for direct incorporation are highly concentrated and are used in excess, thus producing a bright color. Once the direct incorporation RT reaction is complete the excess dye is removed and the purified cDNA, when resuspended in hybridization buffer, does not have much color. Similarly, our Vial 1 does not have much color because we have purified the excess dye from the 3DNA reagent after the synthesis process. If you were to read a couple of microliters in a fluorometer at the proper wavelength, you would detect signal. Alternatively, you can spot 1 ul of a 1:5 dilution of the Vial 1 reagent on a microscope slide and scan it in your scanner to see signal.
Which scanners are compatible with your product?
Based on customer feedback, all commercially available scanners are compatible with our product. The intensity of the signal will vary from scanner to scanner because of differences between the hardware (lasers, filters, etc.) used by each manufacture. You should consult the "User's Guide" for your scanner for laser and PMT settings prior to scanning.
What are the differences between the Submicro and Submicro EX kits?
There are two primary differences between the Submicro and Submicro EX kits, the primer concentration and the use of a spin column to remove excess primer. The Submicro protocol has a lower concentration of RT primer (0.067 pmole/ul) and does not require the use of a spin column to remove excess primer prior to use on the array. Because of the lower primer concentration, the Submicro protocol is limited to total RNA samples and should not be used with poly A RNA.
The Submicro EX protocol has a 5 pmole/ul RT primer concentration and requires a spin column step to remove excess RT Primer after the cDNA synthesis when following the standard kit protocol. The Submicro EX kit can be used for either total RNA or poly A RNA. The reason the Submicro EX protocol is more appropriate for poly A RNA is that it has the higher primer concentration. Excess primer is needed since the oligo dT used in the poly A purification media sometimes leaches off into the poly A RNA preparation, thus competing with the RT primer. The higher concentration of the RT primer in the Submicro EX kit alleviates this problem.
Another difference between the kits is that the Submicro EX kit offers more flexibility than the standard Submicro kit. We have added additional protocols, located in Appendices A and B, for high throughput and ease of use. Both adaptations require additional RNA input but eliminate the need to ethanol precipitate (Appendix A) or spin column purify and ethanol precipitate (Appendix B), thus simplifying the protocol considerably
Can I use your kit for all species of RNA?
Because the kits
provide an oligo dT based primer, they can be used "as is" only with
samples containing poly A mRNA. They cannot be used with mRNA that does
not contain a poly A tail. If you are using RNA samples from organisms
that do not produce polyadenylated RNA, such as bacteria, and want to
use the Submicro kit, you can design message-specific primers for the
genes that interest you. Each primer oligonucleotide should contain
the published capture sequence (see protocol) located 5' to the sequence-specific
primer. Refer to the diagram below.
Custom Oligonucleotide Primer Sequence:
5' - Genisphere capture sequence - gene specific primer - 3'
All of the synthetic primers to be used should be combined to a final
concentration of 0.067 pmole/ul for the Submicro kit or 5 pmole/ul for
the Submicro EX kit. You should then follow the appropriate kit protocol
as usual.
Can I use the Submicro protocol with a Submicro EX kit, if I dilute the primer?
Yes. Dilute the primer 1/75 in DEPC treated water and follow the standard Submicro protocol using 3 ul of primer for the RT reaction.
Can I scale up the Submicro protocol?
Yes. However, you must increase the amount of primer in the RT reaction. Depending on the requirements of your assay, it may be best to work from the Submicro EX Appendix B protocol.
I currently use SuperScript II enzyme. Can I use my enzyme or do I have to use the one in your kit?
You are not required
to use our enzyme. Our enzyme will work with most RNA samples. However,
because each tissue sample and RNA extraction protocol is different,
some enzymes may work better than others with certain RNA samples. Rule
of thumb: If your enzyme works with a particular RNA sample and the
enzyme in the Submicro kit does not, then try substituting your enzyme
for the one contained in the kit.
Please note that we also sell the following kits that do not include
the enzyme:
Submicro: A100731V12, A100736V12, A100741V12, A100746V12
Submicro EX: A100732V12, A100737V12, A100742V12, A100747V12
What is the purpose of Vial 8 (anti-fade reagent) in your kit? Do I have to use it?
The anti-fade reagent in Vial 8 helps to slow down the oxidation rate of the fluorescent dyes which results in fading of the dye over time, and possibly during hybridization. This fading is especially prominent for the Cy5 dye. Not all users need to add the anti-fade reagent. If your Cy3 and Cy5 are not fading or are of similar intensity, then you may not need to add the anti-fade reagent.
Your protocol is designed for cDNA arrays. Can I use your product on oligo arrays?
Genisphere has a new kit called Submicro Oligo specifically designed for use on oligonucleotide arrays. It can be used with either total or poly (A)+ RNA and requires the same sample size as do the Submicro and Submicro EX kits.
How much RNA should I use with your kit?
While we recommend 1-5 ug of total RNA, this may vary depending on your RNA sample and the sensitivity requirements of your assay. We recommend that you start with 3-4 ug of total RNA or 100-200 ug of poly A, and adjust based on the results you obtain.
Should I treat my RNA sample with DNAse?
This will depend
on the RNA sample. Prior to treating your sample with DNAse you should
check your sample via spectophotometer; 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. Your
RNA sample should be at ~23 kB and the genomic DNA will be at ~7kB.
If you have genomic DNA contamination we do suggest that you treat your
RNA sample with DNAse. If you do, you must then completely eliminate
the DNase before making your cDNA, since DNAse will destroy your cDNA.
What is the best method to follow to purify my RNA for use with your kit?
Many different protocols, as well as commercially available kits from Qiagen, Ambion, and Life Technologies, have been used successfully together with Submicro kits. However, since RNA easily degrades and may contain genomic DNA which will affect the active concentration of your RNA, all samples should be checked by the methods described above and in our protocols.
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