Microarray Basics Support—Getting Started
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We don’t recommend glycogen as a carrier since it is often contaminated with nucleic acids, sometimes has RNases, and can interfere with enzymes.
We do not avoid globin through primer design for the GeneChip™ microarrays. All our assays label and/or amplify globin RNA and the amplified globins hybridize and generate array signals. However, our WT assays generate DNA as hybridization targets, which allow for the generation of strong signals of target transcripts even in the presence of globins.
Our array biochemistry has been tailored to minimize the amplification of rRNA. We cannot disclose the specific mechanisms that are used. Additionally, cross-hybridization signal is reduced through our development of assay conditions that take advantage of the thermodynamic differences between labeled RNA targets and labeled cDNA targets that are hybridized to microarrays with attached DNA probes.
While mRNA should yield good results, we have not validated the product for use outside of total RNA. If you wanted to use mRNA, some optimization will have to done to determine the correct amount of input material.
Axiom™ arrays formerly required a total of 200ng of gDNA per sample, with the exception of the Axiom™ Genome-Wide Pan-African Array set, which requires a total of 300ng of gDNA per sample (100ng per array in the set). As of 2016, there are new guidelines for sample input. All human Axiom™ arrays (except the Axiom™ Genome-Wide Pan-African Array Set) require a total of 100ng. The Axiom™ Genome-Wide Pan- African Array Set still requires a total of 300ng, or 100ng per array. Diploid plants and animals require 150ng per array and polyploid plants and animals require 200ng per array. For Axiom™ Microbiome Arrays, a total of 50ng of gDNA or 17.5 μL of cDNA reaction + 2.5 μL reduced TE buffer starting material is required per array. Please refer to the Axiom 2.0 gDNA sample preparation QRC for more details. Starting DNA must be double-stranded for the purpose of accurate concentration determination. gDNA must be of high purity. DNA should be free of DNA polymerase inhibitors.
Examples of inhibitors include high concentrations of heme (from blood) and high concentrations of chelating agents (i.e., EDTA). The gDNA extraction/ purification method should render DNA that is generally salt-free because high concentrations of particular salts can also inhibit enzyme reactions. DNA purity is indicated by OD260/OD280 and OD260/ OD230 ratios. The OD260/OD280 ratio should be between 1.8 and 2.0 and the OD260/OD230 ratio should be greater than 1.5. We recommend that DNA samples that do not meet these criteria be cleaned up as described under Genomic DNA Cleanup in the Axiom user guide. DNA must not be degraded. The approximate average size of gDNA may be assessed on a 1% agarose gel using an appropriate size standard control. Approximately 90% of the DNA must be greater than 10 Kb in size. Control DNA can be run on the same gel for side-by-side comparison.
Human fecal samples or cDNA samples made from purified RNA have been validated.
DNA was extracted from fecal samples using the PowerSoil™ DNA Isolation Kit from MoBio (Cat. No. 12888-50 or 12888-100).
50ng of input mass is optimal.
The genomic DNA (gDNA) sample must be double-stranded, not degraded and free of any contaminants (e.g., PCR inhibitors and other human/non-human gDNA). The recommended starting amount is 250 ng (5 uL with 50 ng/uL) dissolved in low EDTA TE buffer. High EDTA concentration may negatively impact the downstream enzymatic reactions. It is recommended to run the gDNA samples on a 0.8-1% agarose gel for side-by side comparison with a control DNA (included in the kit). High quality genomic DNA will run as a major band at approximately 10-20 kb on the gel. For more detailed information please refer to chapter 4 Genomic DNA General Requirements in the CytoScan™ Assay User Manual (using the Affymterix™ CytoScan™ Assay Kit, Cat. No. 901808).
All catalog GeneChip™ expression arrays are designed to have a minimum sensitivity of 1:100,000. This concentration ratio corresponds roughly to a few copies of transcript per cell, or an approximate 1.5 pM concentration.
Assuming that there is no RNase contamination, a cRNA hybridization cocktail can be stored for at least one year at –80°C with no loss of sensitivity. The fact that the cRNA has been fragmented prior to the first array hybridization reduces the risk of additional subsequent degradation.
GeneChip™ expression arrays may be stored for up to 16 hours, at 4°C, protected from light, prior to scanning with no noticeable loss of signal intensity. To avoid condensation while scanning, allow the arrays to warm to room temperature before the scan.
In addition to the conventional probe sets designed to be within the most 3' 600 bp of a transcript, additional probe sets in the 5' region and middle portion (M) of the transcript have also been selected for certain housekeeping genes, including GAPDH and Actin. Signal intensity ratio of the 3' probe set over the 5' probe set is often referred to as the 3'/5' ratio. This ratio gives an indication of the integrity of your starting RNA, efficiency of first strand cDNA synthesis, and/or in vitro transcription of cRNA. The signal of each probe set reflects the sequence of the probes and their hybridization properties. A 1:1 molar ratio of the 3' to 5' transcript regions will not necessarily give a signal ratio of 1.
There is no single threshold cutoff to assess sample quality for all of the diverse organisms and tissues. This is due to the presence of different isoforms of these house-keeping genes and their different expression patterns in various tissues and organisms. Although we routinely refer to a threshold ratio of less than 3 for the most common tissues, such as mammalian liver and brain, this may not be applicable to all situations. It may be more appropriate to document the 3'/5' ratios within a particular study and flag the results that deviate, therefore representing an unusual sample that deserves further investigation.
The four transcripts are added to the hybridization cocktail at staggered concentrations. At 1.5 pM, bioB is at the detection limit for most expression arrays and is anticipated to be called Present at least 70% of the time. In contrast, the other controls should be called Present all of the time, with increasing Signal values (bioC, bioD, and cre, respectively). Absent calls, or relatively low Signal values, indicate a potential problem with the hybridization reaction or subsequent washing and staining steps. Check to see if the hybridization cocktail was prepared correctly, if the recommended hybridization temperature and Fluidics Protocol were used, and make sure the SAPE staining solution did not deteriorate. Other than qualitative calls and Signal values, the 3'/5' ratio data for these controls are not as informative since they do not relate to the quality of the samples and data.
For Research Use Only. Not for use in diagnostic procedures.