GeneChip™ miRNA 4.0 Assay, 30 samples - FAQs

Probes for snoRNA, scaRNA, and hairpins are selected to maximize probe response to target concentration in the sample while minimizing cross-hybridization to other potential targets in the sample. In order to minimize cross-hybridization, potential targets are inferred from the landscape of known sequences from the input dataset and used in a filtering process called pruning which penalizes probe candidates that may cross-hybridize to unwanted targets. As the landscape of known sequences improves, we can improve our pruning set to avoid probe candidates previously thought to be unique. An improved pruning set will reduce the chances a probe will cross-hybridize to unwanted target, improving the probe set representation of the intended target. As a result of the improved probe selection, few probe sets are required to represent the same number of sequences.

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Probes on the array detect sense target.

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At least 5 miRs or SnoRNAs should be used to normalize:

RU44, RU48, and/or U6 microRNAs that are not changed among your samples, and are at least 5X over background, according to your microarrays. These miRs might include miR 15,16, 17, or let 7a, let 7b, let7c

All 5 (or more) of these RNAs should not show any change among the samples. Average some or all of these to get the normalization factor, and apply to your qRT-PCR data.

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qPCR is not yet the gold standard for miRNA validation. Unlike mRNA validation, in which the amplicon is already present in the sample, miRNA qPCR requires the amplicon to be synthesized by combining the sample with either a specially designed hairpin molecule, adding a 3' polyA tail, or some other manipulation of the microRNA sample. The amplicon-building process will be different from sample to sample and will result in variability in the PCR results. However, it is still very important to validate the array results with another method, like PCR. The trends in up and down regulation should match in direction, even if they do not match in magnitude.

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F Sato. Intra-Platform Repeatability and Inter-Platform Comparibility of microRNA microarray technology. PLoS ONE May 2009, volume 4, issue 5, e5540.
D Sarkar. Quality Assessment and data analysis for microRNA expression arrays. Nucleic Acids Research 2009, vol. 37, no. 2, e17 (doi: 10.1093/ner/gkn932).

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Most researchers eliminate the irrelevant species from analysis, because the extra data can be cumbersome. However, looking at multi-species probes may provide an increased confidence in the data. We typically observe a clustering of cross-species miRs that are identical, or nearly identical.

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We do not have a specific recommendation. A high-quality RNA sample will have a ratio of at least 1.95. However, if microRNAs are present in samples with lower 260:280 ratios, these samples can still be used.

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DNAse treatment is optional. It is not necessary for RNA samples that have trace amounts of genomic DNA contamination, but it may be beneficial for RNA samples that are highly contaminated with genomic DNA, to more accurately quantitate the RNA. After treating your RNA with DNAse, it is essential that the DNase be inactivated completely before proceeding with the FlashTag procedure, to prevent degradation of the FlashTag reagent (Vial 5). A variety of RNA purification columns/kits may be used to inactivate the DNase. Inactivation of the DNase by high temperature may not completely inactivate the enzyme.

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To accurately determine the concentration of the RNA sample, we recommend Quant-iT RiboGreen RNA Assay Kit (Cat. No. R11490) or the NanoDrop ND-1000 Spectrophotometer.

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Either total RNA or low melecular weight (LMW) RNA can be labeled with FlashTag Biotin HSR. Using total RNA can save time and money, and prevent sample loss.

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Any kit for purification of total RNA or LMW (Low Molecular Weight) RNA will be compatible with FlashTag Biotin HSR. Elute or resuspend the RNA in nuclease-free water. Ensure that the purification method retains low molecular weight species. Some commercial products that have been tested successfully with FlashTag Biotin HSR include:

mirVana miRNA Isolation Kit
RecoverAll Total Nucleic Acid Isolation Kit for FFPE
PureLink miRNA Isolation Kit
TRIzol reagent (total RNA only) with additional overnight -20 degrees C precipitation step during isopropanol precipitation.

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