miRNA Profiling Support—Troubleshooting

Yes, store at –20°C for up to two weeks.

No.

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

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

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

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.

Affymetrix suggests running the ELOSA for every labeled sample. However, if the ELOSA QC Assay is not performed, it is recommended that 2 uL of biotin-labeled sample be saved until the array QC is complete. The labeled sample may be stored at –20°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.

Yes.

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

No, because FlashTag™ Biotin 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.

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

The most common cause of signal in negative control ELOSA wells is the source of BSA. We highly recommend BSA from Sigma, catalog number 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.

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.

• 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

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.

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).

We 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

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

We recommend maintaining consistency in array processing protocols to minimize introduction of variability. Our recommended hybridization time for Affymetrix™ miRNA Array Strip is 20 hours. Affymetrix miRNA Array Strip should be hybridized no less than 20 hours and no longer than 24 hours. We strongly encourage maintaining consistent hybridization times to avoid false positives with respect to differential expression due to hybridization time.

We recommend storing the hybridized array at 4° C in Wash A Buffer for no longer than 4 hours prior to starting the wash/stain protocol. Equilibrate array strip to room temperature before proceeding.

The arrays may be stored at 4° C in Array Holding Buffer for up to 24 hours prior to scanning with negligible impact on data. Equilibrate array strip to room temperature before scanning.

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.