Table 2.
Probes for miRNA ISH Categorized on the Basis of Their Performance
| Type 1a |
Type 1b |
Type 2 |
Type 3 |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Calculation | Data | miR-9* | miR-130a | miR-138 | miR-195 | let-7b | miR-124 | miR-205 | miR-1 | |
| Probe comparison | (2OMe + LNA [+])/(DNA + LNA [+]) | Intensity | = | ↑ | ↑ | ↑ | = | = | = | ↓ |
| SNR | = | ↑ | = | ↑ | ↑ | = | = | ↓ | ||
| Blocking agent effect | (DNA + LNA [–])/(DNA + LNA [+]) | Intensity | = | = | ↓ | = | ↓ | = | ↓ | = |
| SNR | = | = | = | = | = | = | ↓ | = | ||
| (2OMe + LNA [–])/(2OMe + LNA [+]) | Intensity | ↑ | ↑ | ↑ | = | = | = | = | = | |
| SNR | ↑ | ↑ | ↑ | = | = | = | = | = | ||
| New method vs gold-standard | (2OMe + LNA [–])/(DNA + LNA [+]) | Intensity | ↑ | ↑ | ↑ | ↑ | ↑ | = | = | ↓ |
| SNR | = | ↑ | ↑ | ↑ | ↑ | = | = | = | ||
Three criteria were evaluated: the effect of changing probe from DNA + locked nucleic acid (LNA) to 2OMe + LNA with yeast RNA (yRNA) in the hybridization buffer, the effect of 50 µg/ml yRNA in the hybridization buffer [+] or without yRNA in the hybridization buffer [–], and the direct comparison between the proposed new method, using 2OMe + LNA probes without yRNA in the hybridization buffer, and the hitherto gold-standard method, using DNA + LNA probe with yRNA in the hybridization buffer. The performance was evaluated by calculating the fold changes for a specific effect and indicating this with ↑ if the hybridization assay was improved by 50% or more, with ↓ if it was impaired by 50% or more, or with = if the measured effect was in between these borderlines (i.e., ±50%). A conservative cutoff of 50% was used to disregard variation in intensity caused by using serial sections, performing manual handling of sections, or from imaging, segmentation, and quantification analysis.