FlashTag™ Biotin HSR RNA Labeling Kits, 30 reactions - FAQs

The ERCC controls are not tiled onto GeneChip miRNA arrays because they would not work with the miRNA array/assay system. The FlashTag Biotin HSR RNA Labeling Kits used to label miRNAs is optimized for shortRNAs. Even though longer mRNAs like the ERCC controls will be labeled, the HWS (hyb/wash/stain) conditions are not optimal and consequently, the longer mRNAs typically do not get detected.

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Due to certain miRNAs being conserved across species, there may be multiple copies of the same probe across the miRNA arrays. However, sample processing with the FlashTag Biotin HSR RNA Labeling Kits ensures that there is excess target compared to the probes on the array. Consequently, there is enough sample available to hybridize onto all the replicate probes.

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In order to perform RNA isolation, any of our kits for purification of total RNA or LMW RNA will be compatible with the FlashTag Biotin HSR RNA Labeling Kits.We also recommend that you elute or resuspend the RNA in nuclease-free water to ensure that the purification method retains low mole.

Reverse transcribed miRNA would not work because it would be the wrong sense (antisense, while our probes interrogate sense RNA). Amplified miRNA should work, but the miRNAs are very short. Therefore, adapters may need to be ligated to them or tailing may be needed to get enough runway to complete amplification. It is unknown if any extra sequence may interfere with the hybridization.

If you are using a FlashTag Biotin HSR RNA Labeling Kit for the GeneChip miRNA 3.1 Array Strip, we recommend staying within the range of 130-1000 ng input total RNA.

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Unfortunately, no, we do not recommend using the mRNeasy kit by Qiagen because it will not bind any fragments shorter than 200 bp.

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You should expect high signal and fold-change concordance for transcripts with identical probes on the miRNA 4.0 and miRNA 4.1 Arrays and the miRNA 3.0 and miRNA 3.1 Arrays. The snoRNA, scaRNA, and precursor miRNA probe sets were reselected, and for some probe sets in these categories, the probes are not identical between the two array designs. Because the probes selected may be different, one may observe changes in the probe set signal summary for a given target. This is possible as different probe sequences will have varying affinity to the target and may show a higher or lower absolute probe signal depending on the GC content of the probes. However, fold changes are typically well-correlated to the legacy probe set unless there is cross-hybridization in the legacy probe set. Our expectation is that more unique and specific probes result in more specific hybridization to the intended target, and therefore a better performing probe set. Target preparation replicates of human brain and lung samples were pooled and hybridized to four miRNA 3.0 Arrays and four miRNA 4.0 Arrays. The Pearson product moment correlation was calculated from the median signal of matched probe sets and from the median fold change of detected matched probe sets on the miRNA 3.0 Array compared to the miRNA 4.0 Array. Probe sets were defined as detected if the median DABG p-value of the four miRNA 3.0 Array brain replicates was less than 0.06. Two subsets of matched probe sets were used for miRNA 3.0 Array to miRNA 4.0 Array signal and fold change correlation: All human probe sets including those that do not necessarily share identical probe sequences and human probe sets for which the probe sequences are identical on both the miRNA 3.0 and miRNA 4.0 Arrays.

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GeneChip Hybridization Control Kit 30 reactions
GeneTitan Hybridization, Wash, and Stain Kit for miRNA Array Plates 96 reactions
GeneTitan Hybridization Module for miRNA Plates 96 reactions
Affymetrix FlashTag Biotin HSR RNA Labeling Kit 10 reactions
Affymetrix FlashTag Biotin HSR RNA Labeling Kit 30 reactions

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Affymetrix miRNA Array Plate provides a similar dynamic range to GeneChip miRNA Array.

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When evaluating non-control probe sets in common to both arrays, we observed very high signal and fold-change correlation for GeneChip miRNA Cartridge Arrays compared to Affymetrix miRNA Array Plates (Pearson correlation coefficients of 0.97-0.98 for signal correlation and Pearson correlation coefficients of 0.96-0.98 for fold-change correlation).

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Array Type: Software
GeneChip miRNA 1.0 Array: miRNA QC Tool Version 1.0.33.0
GeneChip miRNA 2.0 Array: miRNA QC Tool Version 1.1.1.0/Expression Console 1.2 or higher
GeneChip miRNA 3.0 Array: Expression Console 1.2 or higher

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For the GeneChip miRNA Array, please contact Technical Support for the library file. For GeneChip miRNA 2.0, miRNA 3.0, and miRNA 4.0 Arrays, the library files can be downloaded from here (https://www.thermofisher.com/us/en/home/life-science/microarray-analysis/microarray-data-analysis/genechip-array-library-files.html)

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Array Type: Fluidics Station 450 Protocol
400/169 Format Arrays (miRNA 1.0 and 2.0 Arrays): FS450_0003
100 Format Arrays (miRNA 3.0 Array): FS450_0002

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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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We recommend maintaining consistency in array processing protocols to minimize introduction of variability. Our recommended hybridization time for Thermo Fisher Scientific miRNA Array Strip is 20 hrs.

Thermo Fisher Scientific miRNA Array Strip should be hybridized no less than 20 hrs and no longer than 24 hrs. We strongly encourage maintaining consistent hybridization times to avoid false positives with respect to differential expression due to hybridization time.

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GeneChip miRNA Array 17 +/- 1 hr
Thermo Fisher Scientific miRNA Array Plates 17 +/- 1 hr
Thermo Fisher Scientific miRNA Array Strips 20 +/- 1 hr


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GeneChip miRNA Array 130 µL of hybridization cocktail
Thermo Fisher Scientific miRNA Array Plates 120 µL of hybridization cocktail
Thermo Fisher Scientific miRNA Array Strips 120 µL of hybridization cocktail

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You can get the list by downloading the annotation files that will be available on the website.

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The arrays should be stored at 2-8 degrees C.

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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.

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The following hybridization temperatures should be used with each version of ThermoFisher Scientific miRNA Array:

- GeneChip miRNA Arrays 48 degrees C

- Thermo Fisher Scientific miRNA Array Plates 48 degrees C

- Thermo Fisher Scientific miRNA Array Strips 48 degrees C

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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

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We provideTranscriptome Analysis Console Software for normalization, summarization,  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.

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Access the library files on www.thermofisher.com/us/en/home/life-science/microarray-analysis/microarray-data-analysis/genechip-array-library-files.html.

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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, ThermoFisher Scientific miRNA Array Strip, and Thermofisher Scientific 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.

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No, Partek Express and Partek Genomics Suite do not use the .ps file when analyzing data. We will notify you when this has been resolved.

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Yes, Partek Express version 1.12.1121 supports the analysis of miRNA Array Strips.

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IMPORTANT: 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.

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Affymetrix only supports 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 through Affymetrix using Cat. No.s 901910 for the 10- reaction kit and 901911 for the 30-reaction kit.

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The miRNA 4.0/4.1 annotations contain additional data fields to help ease the integration and correlation of small non-coding RNA data contained on Affymetrix miRNA 4.0 and 4.1 Arrays (and newer) with the content from our other expression products such as GeneChip Human Genome U133 Plus 2.0 Array, GeneChip Human Gene 2.0 ST Array, and GeneChip Human Transcriptome Array 2.0. These additional fields include:

- Genomic context (where the miRNA is located in the genome)
- Clustered miRNA (miRNA that are located 10 KB from one another)
- Predicted miRNA targets
- Validated miRNA targets

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We have changed the naming convention for probe sets on our miRNA arrays to be consistent with other array designs. Probe set IDs on Affymetrix miRNA 4.0 and 4.1 Arrays (and newer) will be a unique number. The probe set name will have the accession number and an Affymetrix suffix (e.g., “_st”). While the transcript ID is no longer contained in the probe set name, it is included in the annotation file.

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No, the CEL files from GeneChip miRNA 3.0 Cartridge Array, Affymetrix miRNA 3.1 Array Strip, and Affymetrix miRNA 3.1 Array Plates are not compatible.

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The arrays have the exact same design and contain the same number of probes and probe sets.

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The recommended hybridization time is 17 ± 1 hour.

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120 µL of hybridization cocktail.

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You can get the list by downloading the annotation files that will be available on the website.

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The array plates should be stored at 2-8 degrees C.

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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 by ThermoFisher Scientific 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.

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The CEL files generated from this array is compatible with  Transcriptome Analysis Console and can be used for normalization, signal summarization and differential expression analysis of your experimental data.

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.

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Access the library files on www.thermofisher.com/us/en/home/life-science/microarray-analysis/microarray-data-analysis/genechip-array-library-files.html.

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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 4.0 Array, Affymetrix miRNA 4.1 Array Strip, and Affymetrix miRNA 4.1 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.

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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.

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We only support use of the Affymetrix FlashTag Biotin HSR RNA Labeling Kit for preparing targets to be applied to GeneChip miRNA Array .

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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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In Partek, the limit of detection = 2 Standard Deviations over background. In miRNA QC Tool, click 2X to subtract the background. At this point, anything above background (as defined by the project description table) is significant, if the p-value is also significant (as determined by the researcher).

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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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Less total RNA was loaded on those arrays
Those RNA samples were a little degraded
Those RNA samples were not human (even mouse would have lower signal intensity - homology is not perfect to human)
Those RNA samples were significantly enriched for 22mer / microRNA, removing the 5.8S rRNA which is around a 150mer

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No, 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.

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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, 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.

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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.

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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.

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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.

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Please see the FlashTag Biotin HSR RNA Labeling Kit User Manual for details.

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No, we recommend using AGCC Software.

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The most common cause of signal in negative control ELOSA wells is the source of BSA. We highly recommend BSA from Sigma (Cat. No. A3294). Always use fresh pipet tips to add reagents to each individual well of the ELSOA plate. This will avoid carryover from one well to the next.

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Store all buffers that contain BSA at either 4 degrees C or -20 degrees C. All materials (tubes, tips, etc.) should be nuclease-free, and all reagents should be prepared with nuclease-free components.

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No, because FlashTag reactions are not purified, the same amount of biotin ligation mix (Vial 5) would be detected by the gel-shift assay, whether the labeling was successful or failed.

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Mild shaking (50-300 RPM) is acceptable during sample hybridization and all other steps of the ELOSA, but is not required.

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Yes, this is acceptable.

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Affymetrix suggests running the ELOSA for every labeled sample. However, if the ELOSA QC Assay is not performed, it is recommended that 2 µL of biotin-labeled sample be saved until the array QC is complete. The labeled sample may be stored at -20 degrees C for up to two weeks prior to running the ELOSA. Retaining this sample will enable one the ability to troubleshoot possible target preparation issues, if needed.

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Successful biotin labeling is verified via a simple colorimetric ELOSA assay through the hybridization of the biotin-labeled RNA Spike Control Oligos (Vial 8) to complementary ELOSA Spotting Oligos (Vial 9) immobilized onto microtiter plate wells. Refer to Appendix A of the manual.

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No, we recommend using the Affymetrix GeneChip Hybridization, Wash and Stain Kit (Cat. No. 900720).

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We recommend using the Array Holding Buffer supplied with the Affymetrix GeneChip Hybridization, Wash and Stain Kit.

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Yes, and if necessary, add more volume of 1X hybridization mix prior to re-hybridization. Refer to Appendix B, Array Rehybridization procedure.

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16 to 18 hours.

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The Affymetrix miRNA Arrays do not require pre-hybridization.

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Yes, store at -20 degrees C for up to two weeks.

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

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Yes. PCR plates and thermal cyclers be used for the incubation steps in FlashTag HSR labeling.

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Store the biotin-labeled RNA samples on ice for up to 6 hours, or at -20 degrees C for up to two weeks.

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0.25 ng/µL

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

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RNA oligo 2 at 0.75ng/µL x 2µL = 1.5ng
RNA oligo 23 at 0.75ng/µL x 2µL = 1.5ng
RNA oligo 29 at 0.05ng/µL x 2µL = 0.1ng
Poly(A) RNA oligo 31 at 0.05ng/µL x 2µL = 0.1ng
Poly(A) DNA oligo 36 at 0.05ng/µL x 2µL = 0.1ng

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Vial 8 consists of five oligos which are spiked into the RNA sample prior to FlashTag 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).

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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.

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Vial 3 contains 10mM ATP.

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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

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

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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.

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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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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 product insert.

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Any strand of RNA with a 3'OH will be labeled with FlashTag.

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

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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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