POCDS can offer very substantial further discounts for bulk orders/ special pricing/ package deals on the full range of Eppendorf pipettes (Includes Eppendorf Research plus pipettes: Research Pack starter kits; Research plus single and multi-channel; Xplorer manual and electronic; Easypet pipette boys, Multipette dispensers, Varispenser bottle top dispensers; Stream and Xstream electronic dispensers etc
We have endeavoured to provide Australian catalogue numbers; however it would be prudent to verify any catalogue numbers taken straight from our site with POCD Scientific before using them to place an order.
Biomaster Kit consisting of:
1 Biomaster with continuous volume selection in range from 1-20 Âµl
and 1 box of 96 Mastertips
Surface of a solid slide (e.g. glass, silicon, plastic), on which systematically arranged biomolecular probes like nucleic acids, antibodies, peptides or proteins are immobilized.
When pipetting with the Biomaster, air is drawn into the tip. Is the pipette leaking?
It's unlikely that the pipette seal is faulty. More likely is that the tip seal is faulty. If the pipette is pressed down too hard when attaching tips from the rack, the leakage seal of the piston in the tip can be negatively affected. This is considerably more noticeable with highly viscous solutions than with fluid solutions. In addition, the tips may not be autoclaved.
How do you define detection?
The proof of the signals from the hybridized, radioactive or alternative labeled molecules (->sample / target). By localization of the signals on the microarray the researcher gets the sequence information about the target material. A signal shows that the sequence of the target is complementary to the coupled and immobilized probe sequence spotted on the microarray. The detection method depends upon the type of labeling.
How many arrays do I need for a study?
This depends on the replicates you would like to hybridize with the same RNA (technical replicates). Some parameters influencing the microarray data like the RNA quality and all subsequent steps are better controlled with technical replicates.
This also depends on how tight you would like to control your experimental variance with a reference RNA after RNA preparation (Reference with a two-color labeling competitive on the array, Reference on the 2nd Array on each slide, Reference one per kit, Reference one per lot etc.).
How can I get data for class predictions or bigger studies?
We recommend to use a Reference RNA which should be chosen to have genes expressed in a reasonable way which you would like to study (to get quantitative data and not have a saturated signal or one which is too low), e.g. Stratagene Universal reference RNA (human # 740000; mouse # 740100; rat # 740200).
A Reference RNA is a tool to control better experimental variance after RNA preparation.
Compute ratios of your experimental samples with the reference RNA (do not use the housekeeping genes for normalization in the 2nd step as they are likely to be very different between experimental sample and reference sample).
Then decide if the status (not in linear range, qualitative, quantitative / not the significance values of the ratios (unchanged, significant, highly significant) has to be incorporated somehow in the data analysis (like omitting the data which is not quantitative).
Clustering techniques may then be applied and statistical tests on groups like ANOVA.
Which kind of bar-code is used for microarrays from Eppendorf?
All microarrays from Eppendorf contain a sticker with a barcode â€“ type 128c.
What are DNA microarrays?
"DNA microarrays consist of a large number of DNA molecules that are spotted on modified glass slides, nylon membranes or silicon wafers. A microarray can contain hundreds, thousands, or tens of thousands of genes. Just as microprocessor chips enable computer users to process thousands of bits of information simultaneously, microarrays allow researchers to screen thousands of genes in a single experiment.
The diameter of the spots is between 80 Âµm and 300 Âµm depending on the spotting system."
How should the DualChip microarrays be handled and stored?
DualChip microarrays should be stored at 4Â°C and opened prior to hybridization. The operating environment should be as clean as possible. The DualChip microarrays should be handled from the labeled side (barcode-sticker) only and should never be touched on the window of the frame. After removing the frame, it should be avoided to touch the spotted surface of the slide. Never use a permanent or water resistant marker or any other type of pen on the slide because it may cause interference during the scan process.
What is a probe?
Biomolecule of known identity (e.g. nucleic acid) which is immobilized on the slide. (Cf. conventional blotting methods, where probe = marked molecule, not immobilized). Nomenclature of probes and targets: The first publication dealing with microarray analysis (Schena et al., 1995) uses the term target to describe the immobilized DNA molecule onto the microarray substrate and probe to describe the sample that will be analyzed. This naming is based on the traditional northern blot nomenclature, where the term probe describes the immobilized sample, that will be analyzed. In principle the intention of northern blot analysis are the same as of microarray experiments, that is to measure the expression level of genes. However, the inversion of the physical locations of probes and targets led to some confusion within the microarray community. Therefore, you will always find some researchers, that are using probe to describe the immobilized DNA, whereas others describing with this term the sample, that will be analyzed. To stop this never-ending discussion, the editors of a comprehensive review of microarray technology entitled "The Chipping Forecast", published in Nature Genetics (1999), encouraged their authors to describe the immobilized DNA molecules always as probe and the free nucleic acid as target. Corresponding to this standard nomenclature, you will find this terminology within all Eppendorf writings concerning microarray technology.
What is a sample / target?
Marked biomolecule (e.g. nucleic acid) whose sequence or expression is to be investigated. The sample/target can be marked with a variety of compounds, but is frequently marked with fluorescent dyes. (Cf. conventional blotting methods, where sample = biomolecule immobilized on a carrier).
What is a slide (unspotted support)?
Surface-activated carrier (e.g. slide made of glass or plastic) on which biomolecules (in this case nucleic acids) can be immobilized.
What is a spot?
The region on a microarray which contains biomolecules of the same identity and a defined concentration. A spot is up to 300 Âµm in diameter, depending on the surface parameters and the kind of pins.
What is a DualChip?
The DualChip is the microarray format of Eppendorf. The glass slide contains 2 identical microarrays.
What is included in the DualChip kit for gene expression?
The DualChip kit for gene expression includes 4 seperately sealed slides with two identical arrays per slide. The hybridization chambers are already attached to the slides. The kit includes the hybridization buffers, positive Hybridization controls, Internal Standard spike-ins, and 3 different manuals, specific for the three main laboratory strategies: SIlverquant/ colorimetric; indirect fluorecence, and direct fluorescence.
Based on the manual, the reagents for the RT can be chosen by the user.
Where do I store the components of the kit?
The kit should be stored at +4Â°C. The Internal Standard (2 tubes) should be stored at -80Â°C. After resuspension, the positive hybridization control should be stored at -20Â°C.
Which species do the DualChips cover?
The DualChip is available for human, mouse, and rat.
What kind of DNA is printed on the DualChip microarrays for gene expression?
Each gene on a DualChip microarray is represented by proprietary Xmer probes. These long DNA molecules combine the excellent hybridization efficiency of a cDNA fragment with the ability of an oligonucleotide to distinguish homologous genes. Xmers are perfect for quantitative and sensitive expression profiling experiments where it becomes necessary to study highly homologous genes.
How long are the Xmers on DualChip microarrays?
Can I differentiate between different splice-variants?
It may be, that different splice variants of one gene exist. In that situation, the particular variants which can be detected by the respective Xmer capture probe are given in the gene list provided with each DualChip. For questions regarding specific splice variants, please contact the Eppendorf microarray application support.
Where do I get sequences for real-time PCR primer-design?
The accession numbers for each gene on the DualChip include the version number of sequence. This version number defines the sequence which was used to generate the Xmer probe. NM_004324.1 is the ref-seq-code for the gene BAX, based on the first version. For ABI2, the ref-seq-code NM_005759.3 was used, i.e. the third version of the sequence was used to generate the Xmer sequence.
For specific sequences of Xmer-probes, please contact the microarray application support.
Which RNA should I use for the microarray experiment?
Total RNA or mRNA can be used for the gene expression microarray experiments. Total RNA is recommended.
How should I isolate my RNA?
For the RNA isolation, using a combination of a phenol-containing step (like TrizolÂ® (Invitrogen) or QIAzol (Qiagen)) and a clean-up-column (like Qiagen RNeasy MinElute kit) is recommended.
What amount of RNA do I need?
For the DualChip for gene expression, the amount of total RNA depends on the labeling strategy:
Fluorescence direct labeling: 20-25 Âµg total RNA
Fluorescene indirect biotin labeling: 5-10 Âµg total RNA
Silverquant indirect biotin laboratory: 2-5 Âµg total RNA
using linear RNA-amplification (one round): 10-100 ng total RNA
Can I still perform array analysis if I only have 1 Âµg of total RNA from my sample?
Yes, but you should anticipate weaker signals on the chip.
How should I check the quality of the total RNA?
BioAnalyzer (Agilent): RIN factor > 7
OD260/280 in TE-buffer at least 1.9
denaturating agarose gel (The overall quality of an RNA preparation may be assessed by electrophoresis on a denaturing 1% agarose gel (SamBrook et al., eds. (1989) Molecular cloning â€“ a laboratory manual, 2nd ed. Cold spring Harbor, NY: Cold spring Harbor Laboratory Press). The integrity of the total RNA is assessed by visualization of intact ribosomal RNA bands. For the total RNA from higher eucaryotes, the size of the ribosomal bands should be 1.9 kb for the 18S-RNA and 4.7 kb for the 28S-RNA.)
Concentration 0,35 â€“ 1,6 Âµg/ Âµl (reaction volume: 6 Âµl for RNA)
Can I use random primer to process the reverse transcription?
Not for total RNA as random primers would allow to reverse transcribe the complete amount of RNA molecules. Only the mRNA with PolyA-tail are to be reverse transcribed.
Do I have to use a specific enzyme for the RT-reaction?
No, the Superscript II or III (Invitrogen) are recommended in the manual for optimal sensitivity, but you can use other enzymes as well.
Can Cy3/Cy5 dual labeling be used with DualChip microarrays?
What kind of labeling system can be used?
Colorimetric methods like Silverquant can be used as well as any fluorescent dyes, e.g. Cy3, Cy5, Alexa Fluor.
What kind of controls are included in the DualChip microarray kit?
1. Internal standard mix
- spike-in control, has to be added to the cDNA synthesis reaction
- can be used as positive control for the cDNA synthesis and for normalization of the results
- can be used for normalization of generated ratios
- In the case of two-color analysis it also allows the evaluation of dye bias
2. Pre-labeled positive hybridization controls (biotinylated PCR product)
has to be added to the hybridization solution
- can be used to control hybridization efficiency
3. Positive biotin detection control (probe spotted on the array)
- can be used to control biotin detection efficiency
4. Negative hybridization control (probe spotted on the array)
- to monitor the specificity of the assay
5. Negative detection control (probe spotted on the array)
- to monitor the specificity of the detection process.
How is the high product quality of DualChips assured?
DualChips are guaranteed to have a mean coefficient of variation (CV) in inter-replicates of equal to or less than 10% (3 replicates of each probe per array), an inter-array CV of equal to or less than 15% (2 arrays on one slide) and an inter-slide CV of equal to or less than 20%.
This guarantee is maintained by several steps:
1. General spot control on each array produced
2. Control of the fluorescence background for each batch of arrays produced
3. Control of the inter-slide assay reproducibility for each batch of arrays produced
4. Reagents quality control with each new batch production of the kit components .
Do I need a specific high-end hybridization system for my DualChips?
No, for the DualChips for gene expression, the Thermomixer comfort with the Thermoblock for slides DC is recommended to receive optimal results. There is no need to use expensive hybridization systems. Additionally, the Thermomixer can be used for the RT by using the Thermoblock for 1.5 ml tubes.
How long is the recommended hybridization time?
The optimal hybridization time is overnight (between 12-16h).
What is hybridization?
The process of pairing together complementary strands of nucleic acids. The two strands are called hybrids.
Should I prehybridize?
What is the recommended hybridization temperature?
60 Â°C if using the recommended hybridization buffer. If using other hybridization buffers the optimal temperature might change.
What is the recommended hybridization volume?
When using the Eppendorf hybridization chambers of the DualChip, the recommended volume is 100 Âµl.
What type of hybridization buffer can be used?
60 Â°C if using the recommended hybridization buffer. If using other hybridization buffers the optimal temperature might change.
Which side is the right side to hybridize on?
When slides are placed so that you can read â€śDualChip microarraysâ€ť and the barcode number, the spotted side will be up.
What type of washing buffer is recommended?
We clearly recommend to use the supplied washing buffer.
How can I dry the hybridized DualChip microarrays?
Dry the DualChip microarrays either by centrifugation in a specialized adapter for 5 min at 600 rpm (e. g. CombiSlide Adapter, Eppendorf, used in a Swing-bucket rotor plus Centrifuge 5430 R from Eppendorf) or an adapter for 50 ml-tubes (one slide per tube, barcode sticker on top; 800 rpm 10 min). Drying with a Nitrogen-stream is suboptimal as many nitrogen pipes in older labs are not that clean anymore. There is a risk of receiving small particles on the array.
Which scanner do I need to scan my DualChips?
The Eppendorf microarray slides have the standard slide format of 25 x 75,4 mm. After hybridization, the hybridization frames are removed and the slide can be scanned.
The type of the scanner depends on the labeling strategy:
When using fluorescence labeling, every microarray slide reader is usable like scanners from Tecan, Molecular Devices/ Axon, or Perkin Elmer.
The output format should be a 16-bit *.tif-file.
Which software do I need to perform the quantification of my image?
Quantification means extraction of the signal values out of the image. Normally, the scanner already includes a software to quantify the image-files.
How do I normalize my data?
Eppendorf offers special normalization processes for focused microarrays like the DualChips. For colorimetric-based data, the second module of the Silverquant analysis software enables the user to normalize the DualChip data by using a specific two-step-process. For fluorescence-based data, the evaluation software is recommended.
Can I use the normalization process for other microarrays than Eppendorf DualChips?
No, the normalization process is based on the Internal Standards which are spotted on the DualChip as well as on the selected housekeeping genes.