Table 1.
miRNA probe | Tag | Label | Multiplex | Tissue | Procedure | Ref. |
---|---|---|---|---|---|---|
miR-9, miR-124, miR-125b | DIG | ChromoNBT/BCIP | N/D | Brain | Manual | [51] |
miR-21 | FITC | ChromoDAB | N/D | Colon | Manual | [71] |
miR-150 | DIG | ChromoNBT/BCIP | N/D | Colon, Lymphoid | Manual | [106,118] |
miR-21, miR-155 | DIG | ChromoNBT/BCIP | N/D | Pancreas | Manual | [72] |
Let-7b, miR-205 | DIG | Fluorescent | BreastTMA | Manual | [103] | |
let-7a, let-7b, let-7c, let-7f, let-7g, miR-98 | DIG | ChromoDAB | N/D | BreastTMA | Manual | [107] |
let-7a, miR-21, miR-125b, miR-145, miR-205, miR-451 | FITC | Fluorescent | N/D | BreastTMA, LungTMA |
Manual | [69,142,143] |
miR-21, miR-126, miR-155, miR-182, miR-210 | DIG2X | ChromoNBT/BCIP | N/D | LungTMA | Manual | [82–86] |
miR-146, miR-375 | DIG2X | Fluorescent or ChromoNBT/BCIP | N/D | Lung, Esophagus | Manual; 2-fluoro RNA probes | [87,88] |
miR-200a | DIG2X | ChromoNBT/BCIP | N/D | BreastTMA | Manual | [89] |
miR-21 | DIG2X | ChromoNBT/BCIP | N/D | PancreasTMA | Manual | [90] |
Let-7a, miR-1, miR-21, miR-103, miR-124, miR-126, miR-143, miR-145, miR-205, miR-223 | DIG2X | ChromoNBT/BCIP | N/D | Brain, Breast, Colon | Manual | [77,78,91] |
miR-205 | DIG2X | Fluorescent | ISH/IFplex: 1 RNA + 1 protein |
Prostate | Manual | [92] |
miR-124 | FITC | Fluorescent | ISH/IFplex: 1 RNA + 1 protein |
Brain | Manual; HIER instead of PK | [99] |
let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7glet-7i, miR-1, miR-7a, miR-9, miR-9*, miR-15a, miR-16, miR-21, miR-22, miR-26a, miR-26b, miR-27a, miR-28, miR-29a, miR-30a, miR-30d, miR-99a, miR-99b, miR-101a, miR-122, miR-124, miR-126*, miR-128a, miR-128b, miR-129, miR-130a, miR-133a, miR-132, miR-134, miR-135b, miR-140, miR-143, miR-153, miR-191, miR-370, miR-410, miR-434, miR-5p, miR-467a, miR-497, miR-744, miR-872 | DIG | Fluorescent | IHC/ISHplex: 1 protein + 1 RNA | Mouse tissues | Manual; Post-fixation with EDC |
[79] |
miR-205, miR-375 | FITC6X, Bio6X | Fluorescent | ISHplex: 3 RNAs |
Skin | Manual; Post-fixation with EDC | [81] |
miR-21, miR-34a, miR-92a, miR-205, miR-221 | DIG | Fluorescent | ISH/IHCplex: 1 RNA + 1 protein |
BreastTMA SkinTMA | Manual | [101,102] |
miR-142–5p | DIG | Fluorescent | ISH/IFplex: 1 RNA + 2 proteins |
Brain | Manual; EDC fixation post-HIER | [100] |
miR-1, miR-125b, miR-199a | DIG2X | Fluorescent | ISH/IFplex: 1 RNA + 2 proteins | Heart | Manual | [93] |
miR-21, miR-205 | DIG2X | Fluorescent | ISH/IHCplex: 1 RNA + 1 protein |
Breast, Colon | Semi-automated | [94] |
let-7a, miR-10b, miR-21, miR-24, mir-34a, miR-34c-5p, miR-125b, miR-126, miR-141, miR-143, miR-145, miR-155, miR-196, miR-200b, miR-205, miR-214, miR-221, miR-375, miR-451 | FITC2X, Bio2X, DIG2X, BrdU2X | Fluorescent | ISH/IHCplex: 4 RNAs + 1 protein 2 RNAs + 3 proteins 1 RNA + 4 proteins |
Breast, PancreasFNA, Colon, Lung, Prostate | Semi-automated | [75,95,96] |
miR-21 | DIG2X | ChromoNBT/BCIP | N/D | Colon | Fully- automated | [97,98] |
miR-21, miR-126, miR-145 | DIG2X | ChromoNBT/BCIP | ISH/IHC2plex: 1 RNA + 1 protein |
Mouse and rat tissues | Fully- automated | [80] |
let-7a, miR-10b, miR-21, miR-34a, miR-126, miR-145, miR-155, miR-205, miR-210 | FITC2X | Fluorescent | ISH/IHC4plex: 2 RNAs + 2 proteins 1 RNAs + 3 proteins |
Breast | Fully- automated | [76] |
†This table summarizes main technical features and capabilities of tissue-slide based assays for in situ detection of miRNAs in formalin-fixed paraffin-embedded normal and tumor tissues from various organ sites. Unless otherwise indicated, in situ miRNA detection was performed on whole-tissue sections. From the top to the bottom of the list, the evolution of this assay can be appreciated by the implementation of innovative features and technological advances, from multi-tag probes to fully automated performance.
Bio: Biotin; BrdU: Bromodeoxyuridine; ChromoNBT/BCIP and ChromoDAB: A chromogenic stain with alkaline phosphatase-mediated development of 4-nitroblue tetrazolium and 5-bromo-4-chloro-30-indolyl phosphate substrate or horseradish peroxidase-mediated development 3,3’-diaminobenzidine substrate, respectively; DIG: Digoxigenin; FITC: Fluorescein; FNA: Fine-needle aspirate; IHC: Immunohistochemistry (enzyme conjugated to antibody develops signal); ISH: In situ hybridization; IF: Immunofluorescence (signal directly conjugated to antibody); TMA: Tissue microarray.