miRNA Profiling Support—Getting Started

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:

• Applied Biosystems™ mirVana™ miRNA Isolation Kit
• Applied Biosystems™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE
• Invitrogen™ PureLink™ miRNA Isolation Kit
• Invitrogen™ TRIzol™ reagent (total RNA only) with additional overnight –20°C precipitation step during isopropanol precipitation.

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

To maintain comparability to previous generation arrays, a minimum of 130 ng input is recommended for 100 format arrays.

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™ Biotin 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.

The maximum amount of EDTA in a sample, prior to FlashTag™ Biotin labeling, can be 0.1-1mM. If more EDTA is present, the sample should be desalted/precipitated or purified prior to FlashTag™ Biotin labeling.

Any strand of RNA with a 3’OH will be labeled with FlashTag™ Biotin.

No, specificity is defined by the array itself, using the recommended hybridization conditions. Single nucleotide discrimination is achieved when hybridizing the miRNA Arrays according to the FlashTag™ Biotin product insert.

We don’t 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.

Yes, as long as the FFPE sample contains microRNA. Regardless of the degradation of the FFPE total RNA, use an ATP dilution of 1:500.

Ambion FirstChoice™ Total RNA samples have been used as reference RNA.

The dynamic range of detection on a per molecule basis for any given miRNA is:

0.5 amoles = 300,000 copies

7400 amoles = 4,440,000,000 copies

Vial 3 contains 10mM ATP.

Vial 5 contains a proprietary 3DNA molecule conjugated with biotins. The 3DNA is attached to a poly(T) sequence that facilitates ligation to all poly(A)-tailed RNA molecules.

Vial 8 consists of five oligos which are spiked into the RNA sample prior to FlashTag™ Biotin labeling. These oligos contain controls for the GeneChip™ miRNA array and the ELOSA QC Assay.

• Oligos 2, 23, and 29 are RNA, and confirm poly(A) tailing and ligation.
• Oligo 31 is poly(A) RNA, and confirms ligation.
• Oligo 36 is poly(dA) DNA, and confirms ligation and lack of RNAses in the RNA sample.

The Affymetrix library file lists the following names for these probe sets:

• spike in-control-2 st
• spike in-control-23 st
• spike in-control-29 st
• spike in-control-31 st
• spike in-control-36 st

Each probe set should show >1000 units or 9.96 for log2 signal (signal-background).

RNA oligo 2 @ 0.75ng/uL x 2uL = 1.5ng

RNA oligo 23 @ 0.75ng/uL x 2uL = 1.5ng

RNA oligo 29 @ 0.05ng/uL x 2uL = 0.1ng

Poly(A) RNA oligo 31 @ 0.05ng/uL x 2uL = 0.1ng

Poly(A) DNA oligo 36 @ 0.05ng/uL x 2uL = 0.1ng

Vial 9 contains DNA oligos (complements to RNA oligos 2 & 23) at a concentration of 100 ng/µL. These DNA oligos are used to coat the wells for the ELOSA QC assay.

0.25 ng/uL

Yes.

All materials (tubes, tips, etc.) should be nuclease-free, and all reagents should be prepared with nuclease-free components.

16 to 18 hours.

No, we recommend using AGCC Software.

Please see the FlashTag™ Biotin HSR RNA Labeling Kit (Cat. No. 901910, 901911) User Manual for details.

In miRNA QC Tool software, data is displayed in the window as an “intensity” table. You can re-generate this table by clicking Tables >Intensities. Save the data by clicking on the Save button on the bottom right of the table. This will save the table as a CSV (comma separated value) file that can be imported into third-party software. You can save the data at various “points” in the workflow by checking the “show details” box in the lower left of the intensity data view, and then clicking on the point in the workflow (now displayed in the upper left of the screen) that you want to work from. For example, you can click on raw intensity or background adjustment and save the data at that point to be imported into third-party software.

Yes, the detection is based on the p-value. True does mean present, relative to the p- value. For example: if a miR has low signal and high p-value, it will probably be FALSE. But if a miR has low signal (but still above background) and low p-value, it might be TRUE. Refer to the miRNA QC Tool User’s Guide for more detail.

The p-value is calculated using the 4 probe replicates for each miRNA, and the corresponding variance for each set. Detection means: probe performance/detection using an algorithm and comparing to background.

The normalization control (gi555853 probes) is human 5.8S rRNA. The 5.8S rRNA control probe is the complement to 5.8S rRNA in the sample. If there is any human 5.8S rRNA in the sample, it will be labeled with FlashTag™ Biotin, and hybridize to the miRNA array. When total RNA is titrated, the average signal of these probes also titrates. The rRNA content is variable across samples, and does not necessarily imply performance issues. Signal intensity in rRNA probes is not used for QC as the signals are highly variable between sample types and experimental conditions.

Please see a few references below:

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.

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.

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.

We only support use of the Affymetrix™ FlashTag™ Biotin HSR RNA Labeling Kit for preparing targets to be applied to GeneChip™ miRNA Array, Affymetrix™ miRNA Array Strip, and Affymetrix™ miRNA Array Plate. This product may be ordered using Cat. No. 901910 for the 10- reaction kit and 901911 for the 30-reaction kit.

It is important to know that the hybridization, wash, and stain protocol for Affymetrix™ miRNA Array Strips and Array Plates is different from the 3’ IVT and the Gene ST Array Strips and Array Plates protocols. Additional reagents have been added to these kits to help ensure adequate washing after the stain has been introduced to the array.

• The following instruments and software will be supported for processing GeneChip™ miRNA Arrays:
  • GeneChip™ Scanner 3000 7G and 7G Plus
  • GeneChip™ Fluidics Station 450
  • GeneChip™ Hybridization Oven 640 and 645
  • GeneChip™ Command Console™ Software (AGCC)
• The following instruments and software will be supported for processing Affymetrix™ miRNA Array Strips:
  • GeneAtlas™ System
  • GeneAtlas™ Instrument Control Software (version 1.0.6 or higher)
• The following instruments and software will be supported for processing Affymetrix™ miRNA Array Plates:
  • GeneTitan™ Instrument
• The following analysis software packages are supported for processing Affymetrix™ miRNA Arrays, miRNA Array Strips, and miRNA Array Plates:
  • Expression Console™ Software
  • Transcriptome Analysis Console™ Software

Control Oligo B2 (3 nM) is a pre-labeled DNA spike-in control required for hybridization to the control probes on the array utilized for grid alignment by AGCC. A failure to include Control Oligo B2 in the hybridization cocktail will result in the inability of the software to apply a grid over the scanned image, leading to the unrecoverable loss of sample data from the array. The absence of the Control Oligo B2 also voids the customer's ability to submit an array replacement request.

The Hybridization Controls are high-quality controls for monitoring array hybridization, washing, and staining for reproducible results.

20X Hybridization Controls are composed of a mixture of biotinylated and fragmented cRNA of bioB, bioC, and bioD from E. coli and cre from P1 bacteriophage in staggered concentrations. The premixed controls are ready to be added directly to the hybridization cocktail. Probes for detecting these controls are present on GeneChip™ miRNA Array, Affymetrix™ miRNA Array Strip, and Affymetrix™ miRNA Array Plate.

The 20X Hybridization Controls are spiked into the hybridization cocktail, independent of RNA sample preparation, and are thus used to evaluate sample hybridization efficiency on eukaryotic gene expression arrays. As the 20X Hybridization Controls are used to troubleshoot potential array and array processing issues, a failure to include the 20X Hybridization Controls in the hybridization cocktail will void the customer's ability to submit an array replacement request.

We provide both Expression Console™ Software for normalization and signal summarization of your experimental data and Affymetrix™ Transcriptome Analysis Console Software for differential expression analysis and visualization free of charge. Due to the presence of multiple organisms on the miRNA arrays, the chromosomal location feature in Transcriptome Analysis Console Software will not give accurate results.

For analysis in Transcriptome Analysis Console Software, array type-specific configuration and annotation files are required. Use the “Download Array Type Files” button in the Preferences tab to download and install array type-specific library files.

The following fluidics scripts should be used with each version of GeneChip™ miRNA Array:

• GeneChip™ miRNA Array FS450_0003
• GeneChip™ miRNA 2.0 Array FS450_0003
• GeneChip™ miRNA 3.0 Array FS450_0002
• GeneChip™ miRNA 4.0 Array FS450_0002

The following hybridization temperatures should be used with each version of Affymetrix™ miRNA Array:

• GeneChip™ miRNA Arrays 48˚C
• Affymetrix™ miRNA Array Plates 48˚C
• Affymetrix™ miRNA Array Strips 48˚C

This product has 12 months of shelf life with a minimum shelf life of 3 months. Minimum shelf life is defined as the minimum amount of time from date of order shipment to array expiration date. If users receive products with shorter than expected minimum shelf life, we will replace the products at no cost to the user—subject to terms and conditions.

The arrays should be stored at 2°−8° C.

Minimum is 130 ng; maximum is 1,000 ng total RNA.

GeneChip™ miRNA Array 130 μL of hybridization cocktail

Affymetrix™ miRNA Array Plates 120 μL of hybridization cocktail

Affymetrix™ miRNA Array Strips 120 μL of hybridization cocktail

GeneChip™ miRNA Array 17 +/- 1 hour

Affymetrix™ miRNA Array Plates 17 +/- 1 hour

Affymetrix™ miRNA Array Strips 20 +/- 1 hour

Probes on the array detect sense target.

The only difference is the form factor of the array. The arrays have the exact same design and contain the same number of probes and probe sets.

No, the CEL files from GeneChip™ miRNA Array, Affymetrix™ miRNA Array Strip, and Affymetrix™ miRNA Array Plates are not compatible.