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. 2014 Aug 4;14(7):853–869. doi: 10.1586/14737159.2014.944507

Table 1.

Variations and evolution of tissue slide-based assays for in situ detection of miRNAs†.

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.