Microarray Basics Support—Getting Started

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 (whole transcript) assays generate DNA as hybridization targets, which allow for the generation of strong signals of target transcripts even in the presence of globins. This is in contrast with the IVT (in vitro transcription) assays, where the binding of the generated labeled RNA targets and the array signals are weaker, due to globin interference. Therefore, it is necessary to run a globin reduction when using the IVT assays.

Yes, high amounts of ribosomal RNA (rRNA) can influence the amplification process. Ribosomal RNAs are the most abundant types of RNA present in cells, and they can compete with messenger RNAs (mRNAs), which are usually the targets that bind to the probes on the array, during the reverse transcription and amplification steps. This competition can lead to less efficient amplification of the mRNAs, which can impact the overall quality and reliability of the outcome data.
However, it's important to note that this doesn't necessarily cause "random" primer amplification.
The primers used in these steps are designed to be specific for the mRNAs depending on the assay used and also on the probes on the array, so the presence of excess rRNA shouldn't cause them to bind and amplify random sequences. But it can reduce the efficiency with which they amplify the specific sequences for which the probes are designed for.
As a best practice, depending on the nature of the input, we often recommend that you deplete rRNA from the samples before performing these steps, especially when working with total RNA samples. There are several commercially available kits and methods for rRNA depletion.

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.

gDNA extracted from stool samples has been tested for use with the Axiom Microbiome Array. DNA derived from FFPE (formalin-fixed paraffin-embedded) blocks should not be used with this assay.

Any extraction and purification kit that meets the starting gDNA requirements is acceptable. However, methods that require boiling or strong denaturants should not be used, as the DNA will be rendered single-stranded.

50 ng of gDNA (20 µL at 2.5 ng/µL) is optimal. The gDNA must also be double-stranded, of high purity (OD260/OD280 ratio of 1.8-2.0 and OD260/OD230 ratio greater than 1.5), and not degraded (90% of gDNA greater than 10 kb).

The tested sources of human gDNA used in the CytoScan Assay are blood and cell line samples.
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 µL with 50 ng/µL) dissolved in low EDTA TE buffer. High EDTA concentration may negatively impact the downstream enzymatic reactions. We recommend running 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 the CytoScan Assay User Guide, page 9, Genomic DNA general requirements and recommendations section .

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.

It is an optional step to collect the hybridization cocktail after the first overnight hybridization (16 hrs at 45 degrees C). The hyb cocktail can be collected from the array and transferred to a fresh microcentrifuge tube or well of a 96-well plate to save it. This can be used to re-hybridize new arrays, provided that there is no RNase contamination. The hyb cocktail transfer has to be stored on ice during the procedure and at -20 degrees C for long term storage. For more information please refer to the GeneChip WT PLUS Reagent Kit User Guide, Page 34 in Wash and stain the cartridge arrays section.

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.

β-actin and GAPDH are used to assess RNA sample and assay quality by looking at the 3'/5' ratio. The 3'/5' ratio refers to the signal values of the 3' probe sets for actin and GAPDH being compared to the signal values of the corresponding 5' probe sets. The ratio is generally no more than 3 for the 1-cycle assay. Since the Affymetrix eukaryotic expression assay has an inherent 3' bias, a high 3' to 5' ratio may indicate degraded RNA or inefficient transcription of ds cDNA or biotinylated cRNA.

The 2-cycle assay typically gives higher 3' to 5' ratios than the 1-cycle assay, due to the additional cycle of amplification. There are occasions when the 3' to 5' ratio of one internal control gene is normal, but the 3' to 5' ratio of another internal control gene is high. This discrepancy in 3' to 5' ratios is most likely due to a specific transcript-related or image artifact issue and is not an indication of overall sample and assay quality.

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 50% 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).