Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues
<p>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.</p> "> Figure 2
<p>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 <span class="html-italic">DapB</span> (full form) and a positive control probe <span class="html-italic">PPIB</span> (full form). The <span class="html-italic">PPIB</span> stained well with pancreatic, colorectal and HeLa cells (bottom panel (<b>a</b>–<b>c</b>). 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.</p> "> Figure 3
<p>Paraffin embedded different human cancer tissues that have been Z probe stained for different lncRNAs. (<b>a</b>) Tumor suppressor lncRNA NRON (very low expression, non-coding repressor of NFAT) (<b>i</b>) and oncogenic lncRNA UCA1 (moderately expressed, urothelial cancer associated 1) (<b>ii</b>) stained in colorectal cancer tissue. (<b>b</b>) LncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) stained using specific Z-probes in paraffin embedded (<b>i</b>) colorectal cancer, (<b>ii</b>) breast cancer and (<b>iii</b>) 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.</p> "> Figure 4
<p>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 (<b>a</b>) Stages I–IV CRC tissues show a differential stain for lncRNA MALAT1. Stain intensity correlates with the progression. (<b>b</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.</p> "> Figure 5
<p>LncRNA MALAT1 expression in breast cancer. BioMax TMA BR243w containing 12 normal adjacent tumor (NAT) and 12 invasive breast carcinomas were stained and analyzed. (<b>a</b>) 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>b</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 <span class="html-italic">t</span>-test, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Schematic and Controls
2.2. Assay Validation
2.3. Quantitative Measure of Progression and Invasiveness
3. Discussion
4. Materials and Methods
4.1. Research Involving Human Tissues
4.2. Chromogenic Staining in FFPE Tissues
4.3. Quantitation (ImageJ) Analysis
Summary of ImageJ Analysis
4.4. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Willingham, A.T.; Orth, A.P.; Batalov, S.; Peters, E.C.; Wen, B.G.; Aza-Blanc, P.; Hogenesch, J.B.; Schultz, P.G. A strategy for probing the function of noncoding RNAs finds a repressor of NFAT. Science 2005, 309, 1570–1573. [Google Scholar] [CrossRef] [PubMed]
- Hon, C.C.; Ramilowski, J.A.; Harshbarger, J.; Bertin, N.; Rackham, O.J.; Gough, J.; Denisenko, E.; Schmeier, S.; Poulsen, T.M.; Severin, J.; et al. An atlas of human long non-coding RNAs with accurate 5’ ends. Nature 2017, 543, 199–204. [Google Scholar] [CrossRef] [PubMed]
- Ma, X.; Zhang, W.; Zhang, R.; Li, J.; Li, S.; Ma, Y.; Jin, W.; Wang, K. Overexpressed long noncoding RNA CRNDE with distinct alternatively spliced isoforms in multiple cancers. Front. Med. 2018. [Google Scholar] [CrossRef] [PubMed]
- Gutschner, T.; Richtig, G.; Haemmerle, M.; Pichler, M. From biomarkers to therapeutic targets-the promises and perils of long non-coding RNAs in cancer. Cancer Metastasis Rev. 2018, 37, 83–105. [Google Scholar] [CrossRef] [PubMed]
- Shen, J.; Hodges, T.R.; Song, R.; Gong, Y.; Calin, G.A.; Heimberger, A.B.; Zhao, H. Serum HOTAIR and GAS5 levels as predictors of survival in patients with glioblastoma. Mol. Carcinog. 2018, 57, 137–141. [Google Scholar] [CrossRef] [PubMed]
- Song, W.; Wang, K.; Zhang, R.; Zou, S. Long noncoding RNA MALAT1: A potential novel prognostic biomarkers in cancers based on Meta-analysis. Zhong Nan Da Xue Xue Bao Yi Xue Ban/J. Cent. South Univ. Med. Sci. 2016, 41, 1163–1167. [Google Scholar]
- Kong, H.; Wu, Y.; Zhu, M.; Zhai, C.; Qian, J.; Gao, X.; Wang, S.; Hou, Y.; Lu, S.; Zhu, H. Long non-coding RNAs: Novel prognostic biomarkers for liver metastases in patients with early stage colorectal cancer. Oncotarget 2016, 7, 50428–50436. [Google Scholar] [CrossRef] [PubMed]
- Matos, L.L.; Trufelli, D.C.; de Matos, M.G.; da Silva Pinhal, M.A. Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark. Insights 2010, 5, 9–20. [Google Scholar] [CrossRef] [PubMed]
- Thomsen, R.; Nielsen, P.S.; Jensen, T.H. Dramatically improved RNA in situ hybridization signals using LNA-modified probes. RNA 2005, 11, 1745–1748. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Weiszmann, R.; Hammonds, A.S.; Celniker, S.E. Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos. Nat. Protoc. 2009, 4, 605–618. [Google Scholar] [CrossRef] [PubMed]
- Thisse, C.; Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat. Protoc. 2008, 3, 59–69. [Google Scholar] [CrossRef] [PubMed]
- Zhang, Z.; Weaver, D.L.; Olsen, D.; Peng, Z.; Ashikaga, T.; Evans, M.F. Long non-coding RNA chromogenic in situ hybridisation signal pattern correlation with breast tumour pathology. J. Clin. Pathol. 2016, 69, 76–81. [Google Scholar] [CrossRef] [PubMed]
- Wang, F.; Flanagan, J.; Su, N.; Wang, L.C.; Bui, S.; Nielson, A.; Wu, X.; Vo, H.T.; Ma, X.J.; Luo, Y. RNAscope: A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 2012, 14, 22–29. [Google Scholar] [CrossRef] [PubMed]
- Velcheti, V.; Schalper, K.A.; Carvajal, D.E.; Anagnostou, V.K.; Syrigos, K.N.; Sznol, M.; Herbst, R.S.; Gettinger, S.N.; Chen, L.; Rimm, D.L. Programmed death ligand-1 expression in non-small cell lung cancer. Lab. Investig. J. Tech. Methods Pathol. 2014, 94, 107–116. [Google Scholar] [CrossRef] [PubMed]
- Jacob, F.; Guertler, R.; Naim, S.; Nixdorf, S.; Fedier, A.; Hacker, N.F.; Heinzelmann-Schwarz, V. Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines. PLoS ONE 2013, 8, e59180. [Google Scholar] [CrossRef] [PubMed]
- Taylor, S.; Wakem, M.; Dijkman, G.; Alsarraj, M.; Nguyen, M. A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods 2010, 50, S1–S5. [Google Scholar] [CrossRef] [PubMed]
- Fleige, S.; Pfaffl, M.W. RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Asp. Med. 2006, 27, 126–139. [Google Scholar] [CrossRef] [PubMed]
- Sharma, S.; Findlay, G.M.; Bandukwala, H.S.; Oberdoerffer, S.; Baust, B.; Li, Z.; Schmidt, V.; Hogan, P.G.; Sacks, D.B.; Rao, A. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex. Proc. Natl. Acad. Sci. USA 2011, 108, 11381–11386. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Li, C.; Liang, G.; Yang, S.; Sui, J.; Yao, W.; Shen, X.; Zhang, Y.; Peng, H.; Hong, W.; Xu, S.; et al. Dysregulated lncRNA-UCA1 contributes to the progression of gastric cancer through regulation of the PI3K-Akt-mTOR signaling pathway. Oncotarget 2017, 8, 93476–93491. [Google Scholar] [PubMed]
- Wang, Z.Q.; Cai, Q.; Hu, L.; He, C.Y.; Li, J.F.; Quan, Z.W.; Liu, B.Y.; Li, C.; Zhu, Z.G. Long noncoding RNA UCA1 induced by SP1 promotes cell proliferation via recruiting EZH2 and activating AKT pathway in gastric cancer. Cell Death Dis. 2017, 8, e2839. [Google Scholar] [CrossRef] [PubMed]
- Huang, J.; Zhou, N.; Watabe, K.; Lu, Z.; Wu, F.; Xu, M.; Mo, Y.Y. Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1). Cell Death Dis. 2014, 5, e1008. [Google Scholar] [CrossRef] [PubMed]
- Jadaliha, M.; Zong, X.; Malakar, P.; Ray, T.; Singh, D.K.; Freier, S.M.; Jensen, T.; Prasanth, S.G.; Karni, R.; Ray, P.S.; et al. Functional and prognostic significance of long non-coding RNA MALAT1 as a metastasis driver in ER negative lymph node negative breast cancer. Oncotarget 2016, 7, 40418–40436. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Li, C.; Cui, Y.; Liu, L.F.; Ren, W.B.; Li, Q.Q.; Zhou, X.; Li, Y.L.; Li, Y.; Bai, X.Y.; Zu, X.B. High Expression of Long Noncoding RNA MALAT1 Indicates a Poor Prognosis and Promotes Clinical Progression and Metastasis in Bladder Cancer. Clin. Genitourin. Cancer 2017, 15, 570–576. [Google Scholar] [CrossRef] [PubMed]
- Zheng, H.T.; Shi, D.B.; Wang, Y.W.; Li, X.X.; Xu, Y.; Tripathi, P.; Gu, W.L.; Cai, G.X.; Cai, S.J. High expression of lncRNA MALAT1 suggests a biomarker of poor prognosis in colorectal cancer. Int. J. Clin. Exp. Pathol. 2014, 7, 3174–3181. [Google Scholar] [PubMed]
- Handa, H.; Kuroda, Y.; Kimura, K.; Masuda, Y.; Hattori, H.; Alkebsi, L.; Matsumoto, M.; Kasamatsu, T.; Kobayashi, N.; Tahara, K.I.; et al. Long non-coding RNA MALAT1 is an inducible stress response gene associated with extramedullary spread and poor prognosis of multiple myeloma. Br. J. Haematol. 2017, 179, 449–460. [Google Scholar] [CrossRef] [PubMed]
- Li, Q.; Pan, X.; Wang, X.; Jiao, X.; Zheng, J.; Li, Z.; Huo, Y. Long noncoding RNA MALAT1 promotes cell proliferation through suppressing miR-205 and promoting SMAD4 expression in osteosarcoma. Oncotarget 2017, 8, 106648–106660. [Google Scholar] [CrossRef] [PubMed]
- Xiping, Z.; Bo, C.; Shifeng, Y.; Feijiang, Y.; Hongjian, Y.; Qihui, C.; Binbin, T. Roles of MALAT1 in development and migration of triple negative and Her-2 positive breast cancer. Oncotarget 2018, 9, 2255–2267. [Google Scholar] [CrossRef] [PubMed]
- Huang, N.S.; Chi, Y.Y.; Xue, J.Y.; Liu, M.Y.; Huang, S.; Mo, M.; Zhou, S.L.; Wu, J. Long non-coding RNA metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) interacts with estrogen receptor and predicted poor survival in breast cancer. Oncotarget 2016, 7, 37957–37965. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Soudyab, M.; Iranpour, M.; Ghafouri-Fard, S. The Role of Long Non-Coding RNAs in Breast Cancer. Arch. Iran. Med. 2016, 19, 508–517. [Google Scholar] [PubMed]
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Tripathi, M.K.; Zacheaus, C.; Doxtater, K.; Keramatnia, F.; Gao, C.; Yallapu, M.M.; Jaggi, M.; Chauhan, S.C. Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues. Non-Coding RNA 2018, 4, 20. https://doi.org/10.3390/ncrna4030020
Tripathi MK, Zacheaus C, Doxtater K, Keramatnia F, Gao C, Yallapu MM, Jaggi M, Chauhan SC. Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues. Non-Coding RNA. 2018; 4(3):20. https://doi.org/10.3390/ncrna4030020
Chicago/Turabian StyleTripathi, Manish K., Chidi Zacheaus, Kyle Doxtater, Fatemeh Keramatnia, Cuilan Gao, Murali M. Yallapu, Meena Jaggi, and Subhash C. Chauhan. 2018. "Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues" Non-Coding RNA 4, no. 3: 20. https://doi.org/10.3390/ncrna4030020