Indica
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ISH Quantification

ISH QUANTIFICATION

(Designed for RNAscope®)

 

Product Overview

Indica Labs In-situ Hybridization (ISH) module is recognized as the fastest, most accurate and user friendly analysis tool for quantifying nucleic acid probes across tissues and cell lines. This module measures up to dual chromogenic and/or silver-labeled DNA or RNA ISH probes on a cell-by-cell basis. This allows the user to rapidly contextualize the gene expression profile of every cell across the tissue. HALO ISH analysis is designed to work with single or dual RNA ISH assays including RNAscope™ and supports the H-score protocol for RNAscope as recommended by the manufacturer, ACD Bio.


Watch a webinar from two of our customers discussing how they use and analyze RNAscope assays in their research and download the ACD Bio™ RNAscope Data Analysis Guide by clicking the links here.


Three examples of ISH quantification are shown in the slider above.  In the first example, a kidney section is probed with a single RNA ISH probe (brown) and hematoxylin counterstain. Negative cells are white in the markup image and cells positive for RNA probe are labeled in different shades of red according to their RNAscope score – cells with between 1-3 probe = +1 (light pink), 4-9 spots = +2 (dark pink), 10-13 spots = +3 (light red) and >14 spots = +4 (dark red) as shown in the image below.

The second example is a dual RNA ISH assay in tumor using red and blue probes. Negative cells are identifed using a white overlay mask. Cells positive for the red probe only are shown in different shades of red according to their RNAscope score as described for the single probe assay above. Cells positive for the blue probe only are shown in different shades of green according to their RNAscope score. Cells positive for both markers are in shades of yellow representing the ratio of red: blue probe.

The last example is a dual DNA ISH assay. Like the dual RNA ISH assay, cells containing red probe only are in shades of red, black probe only are in shades of green, cells with both probes are in shades of yellow, and probes with no probe are white.

The module reports the total number of cells, total number of spots, total area of spots, the average number of spots per cell and per unit area of tissue, the average area of spots per cell and per unit area of tissue, as well as the number and percentage of cells with RNAscope score of 0, 1+, 2+, 3+ and 4+. When two probes are present, the module also reports the ratio of probe 1: probe 2. Histograms are automatically generated representing the frequency distribution of probes per cell [Figure 1) and RNAscope cell scoring [Figure 2] as shown below.

Figure 1 shows a distribution curve of probe number on a cell by cell basis, in this example most cells were shown to have between 0-5 copies per cell.

Figure 2. This bar chart shows the numbers of cells that were categorized as 0, 1+, 2+, 3+ and 4+ respectively. The chart shows that a slim majority of cells were positive for RNA probe expression.

The ISH quantification module integrates seamlessly into the HALO™ platform which is compatible with a number of third party systems and  file formats.

Contact info@indicalab.com for product demonstration and pricing information or upload some images for a free trial.

ISH QUANTIFICATION

(Designed for RNAscope®)

Indica Labs In-situ Hybridization (ISH) module is recognized as the fastest, most accurate and user friendly analysis tool for quantifying nucleic acid probes across tissues and cell lines. This module measures up to dual chromogenic and/or silver-labeled DNA or RNA ISH probes on a cell-by-cell basis. This allows the user to rapidly contextualize the gene expression profile of every cell across the tissue. HALO ISH analysis is designed to work with single or dual RNA ISH assays including RNAscope™ and supports the H-score protocol for RNAscope™ recommended by the manufacturer, ACD Bio.


Watch a webinar from two of our customers discussing how they use and analyze RNAscope assays in their research and download the ACD Bio™ RNAscope Data Analysis Guide by clicking the links here.


Three examples of ISH quantification are shown in the slider above.  In the first example, a kidney section is probed with a single RNA ISH probe (brown) and hematoxylin counterstain. Negative cells are white in the markup image and cells positive for RNA probe are labeled in different shades of red according to their RNAscope score – cells with between 1-3 probe = +1 (light pink), 4-9 spots = +2 (dark pink), 10-13 spots = +3 (light red) and >14 spots = +4 (dark red) as shown in the image below.

The second example is a dual RNA ISH assay in tumor using red and blue probes. Negative cells are identifed using a white overlay mask. Cells positive for the red probe only are shown in different shades of red according to their RNAscope score as described for the single probe assay above. Cells positive for the blue probe only are shown in different shades of green according to their RNAscope score. Cells positive for both markers are in shades of yellow representing the ratio of red: blue probe.

The last example is a dual DNA ISH assay. Like the dual RNA ISH assay, cells containing red probe only are in shades of red, black probe only are in shades of green, cells with both probes are in shades of yellow, and probes with no probe are white.

The module reports the total number of cells, total number of spots, total area of spots, the average number of spots per cell and per unit area of tissue, the average area of spots per cell and per unit area of tissue, as well as the number and percentage of cells with RNAscope score of 0, 1+, 2+, 3+ and 4+. When two probes are present, the module also reports the ratio of probe 1: probe 2. Histograms are automatically generated representing the frequency distribution of probes per cell [Figure 1) and RNAscope cell scoring [Figure 2] as shown below.

Figure 1 shows a distribution curve of probe number on a cell by cell basis, in this example most cells were shown to have between 0-5 copies per cell.

Figure 2. This bar chart shows the numbers of cells that were categorized as 0, 1+, 2+, 3+ and 4+ respectively. The chart shows that a slim majority of cells were positive for RNA probe expression.

HALO is compatible with a broad spectrum of image and digital slide formats.  Yours not on the list? Email us your requirements.

Non-proprietary (JPG, TIF)
Nikon (ND2)
3D Histech (MRXS)
Perkin Elmer (QPTIFF, component TIFF)
Olympus (VSI)
Hamamatsu (NDPI, NDPIS)
Aperio (SVS, AFI)
Zeiss (CZI)
Leica (SCN, LIF)
Philips (Requires Philips IMS)

Here are a few publications that cite use of our ISH and FISH modules.  Your publication not on the list?  Drop us an email to let us know about it!

Title Author Year Journal
Breast and pancreatic cancer interrupt IRF8-dependent dendritic cell development to overcome immune surveillance Meyer MA, Baer JM, Knolhoff BL, Nywening TM, Panni RZ, Su X, Weilbaecher KN, Hawkins WG, Ma C, Fields RC, Linehan DC, Challen GA, Faccio R, Aft RL, DeNardo DG 2018 Nature Communications
A randomized, open-label study of the efficacy and safety of AZD4547 monotherapy versus paclitaxel for the treatment of advanced gastric adenocarcinoma with FGFR2 polysomy or gene amplification Van Cutsem E, Bang Y-J,  Mansoor W,  Petty RD, Chao Y, Cunningham D, Ferry DR, N. R. Smith NR, Frewer P, Ratnayake J, Stockman PK, Kilgour E, and Landers D 2017 Annals of Oncology
APNG as a prognostic marker in patients with glioblastoma Fosmark S, Hellwege S, Dahlrot RH, Jensen KL, Derand H, Lohse J, Sørensen MD, Hansen S, Kristensen BW 2017 PLOS One
Differential Cytokine Gene Expression in Granulomas from Lungs and Lymph Nodes of Cattle Experimentally Infected with Aerosolized Mycobacterium bovis Palmer MV, Thacker TC, Waters WR 2016 PLOS One
Fully automated RNAscope in situ hybridization assays for formalin‐fixed paraffin‐embedded cells and tissues Anderson CM, Zhang B, Miller M, Butko E, Wu X, Laver T, Kernag C, Kim J, Luo Y, Lamparski H 2016 Journal of Cellular Biochemistry
G-protein-independent coupling of MC4R to Kir7. 1 in hypothalamic neurons Ghamari-Langroudi M, Digby GJ, Sebag JA, Millhauser GL, Palomino R, Matthews R, Gillyard T, Panaro BL, Tough IR, Cox HM, Denton JS, Cone RD 2015 Nature

Use the form below to upload up to three images for us to analyze using HALO.  If the form does not display, please click here.