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. 2018 Sep 5;4(3):20.
doi: 10.3390/ncrna4030020.

Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues

Manish K Tripathi  1 , Chidi Zacheaus  2 , Kyle Doxtater  3 , Fatemeh Keramatnia  4 , Cuilan Gao  5 , Murali M Yallapu  6 , Meena Jaggi  7 , Subhash C Chauhan  8
Affiliations

Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues

Manish K Tripathi et al. Noncoding RNA. .

Abstract

Formalin-fixed paraffin embedded (FFPE) tissues are a valuable resource for biomarker discovery in order to understand the etiology of different cancers and many other diseases. Proteins are the biomarkers of interest with respect to FFPE tissues as RNA degradation is the major challenge in these tissue samples. Recently, non-protein coding transcripts, long non-coding RNAs (lncRNAs), have gained significant attention due to their important biological actions and potential involvement in cancer. RNA sequencing (RNA-seq) or quantitative reverse transcription-polymerase chain reaction (qRT-PCR) are the only validated methods to evaluate and study lncRNA expression and neither of them provides visual representation as immunohistochemistry (IHC) provides for proteins. We have standardized and are reporting a sensitive Z probe based in situ hybridization method to visually identify and quantify lncRNA in FFPE tissues. This assay is highly sensitive and identifies transcripts visible within different cell types and tumors. We have detected a scarcely expressed tumor suppressor lncRNA NRON (non-coding repressor of nuclear factor of activated T-cells (NFAT)), a moderately expressed oncogenic lncRNA UCA1 (urothelial cancer associated 1), and a highly studied and expressed lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in different cancers. High MALAT1 staining was found in colorectal, breast and pancreatic cancer. Additionally, we have observed an increase in MALAT1 expression in different stages of colorectal cancer.

Keywords: MALAT1; NRON; UCA1; Z probe; breast cancer; colorectal cancer; long noncoding RNA; pancreatic cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
A schematic representation of the Z probe based RNAScope assay for long non-coding RNA (lncRNA) analysis. Starting with Z probes hybridizing with the target sequence creating double ZZs with up to 20 groups side by side. The pre-amplifier then binds to the complementary sequence on the 28-base tail (top of the ZZ). Pre-amplifiers contain multiple binding sites for amplifiers to bind to and the amplifiers also have multiple binding sites for labeled probes to bind. Upon chromogenic stain, the labeled probes fluoresce with a red color.
Figure 2
Figure 2
Validation and optimization of the Z-probe staining. Paraffin embedded, and sectioned, pancreatic cancer tissue, colorectal cancer tissue and HeLa cell pellet were stained with a negative control probe DapB (full form) and a positive control probe PPIB (full form). The PPIB stained well with pancreatic, colorectal and HeLa cells (bottom panel (a–c). 20× (inset) and 80× magnification using CaseViewer 2.2 software (3DHistech Ltd., Budapest, Hungary) scanned and analyzed on Pannoramic 250 Flash III (3DHistech Ltd.). Arrows point at specific staining.
Figure 3
Figure 3
Paraffin embedded different human cancer tissues that have been Z probe stained for different lncRNAs. (a) Tumor suppressor lncRNA NRON (very low expression, non-coding repressor of NFAT) (i) and oncogenic lncRNA UCA1 (moderately expressed, urothelial cancer associated 1) (ii) stained in colorectal cancer tissue. (b) LncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) stained using specific Z-probes in paraffin embedded (i) colorectal cancer, (ii) breast cancer and (iii) pancreatic cancer tissues. 20× (inset) and 80× magnification using CaseViewer 2.2 software, scanned and analyzed on Pannoramic 250 Flash III. Arrows point at specific lncRNA signals.
Figure 4
Figure 4
LncRNA-MALAT in different stages of colorectal cancer (CRC). Paraffin embedded different stages of colorectal cancer tissues were stained for lncRNA MALAT1 and quantitated for staining intensity. 60 CRC tissues, Stage I = 15; Stage II = 16; Stage III = 20; Stage IV = 9, were stained for lncRNA-MALAT1 (a) Stages I–IV CRC tissues show a differential stain for lncRNA MALAT1. Stain intensity correlates with the progression. (b) Quantitation of the lncRNA MALAT1 staining intensity was performed using Image J software. 10× (inset) and 80× magnification using CaseViewer 2.2 software, scanned and analyzed on Pannoramic 250 Flash III. Statistical analysis: One-way ANOVA and Tukey’ multiple comparison tests compare the mean of each column with the mean of other columns.
Figure 5
Figure 5
LncRNA MALAT1 expression in breast cancer. BioMax TMA BR243w containing 12 normal adjacent tumor (NAT) and 12 invasive breast carcinomas were stained and analyzed. (a) Matched breast cancer tissues (NAT vs. Invasive) from two different patients, 1 and 2, were stained for lncRNA MALAT1 using Z probe. The invasive breast cancer tissues have higher staining for lncRNA MALAT1 as compared to normal adjacent tumors. (b) Quantitation of the lncRNA MALAT1 staining intensity was performed using Image J software. 10× (inset) and 80× magnification using CaseViewer 2.2 software, scanned and analyzed on Pannoramic 250 Flash III. Statistical analysis: Unpaired t-test, ** p < 0.01, *** p < 0.001.
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