We thank Dr. Roger Erickson for his support and assistance with preparation of the manuscript.
This work was supported by the National Cancer Institute at the National Institutes of Health
(Grant Number RO1CA177984; RO1CA138642), VA program project on prostate cancer (BX001604).

Formalin-fixed, paraffin-embedded (FFPE) or fresh frozen prostate cancer samples were obtained from the SFVAMC. Samples were from prostate cancer patients who underwent radical prostatectomy at SFVAMC. Written informed consent was obtained from all patients and the study was approved by the UCSF Committee on Human Research. Alternatively, prostate cancer tissues microarrays were procured from commercial sources and used for miRNA analyses by ISH. Clinicopathological and follow up information for analyzed prostate cancer ...

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 65 (1), 5-29 (2015).</li><li>Shen, M. M., Abate-Shen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+prostate+cancer:+new+prospects+for+old+challenges.">Molecular genetics of prostate cancer: new prospects for old challenges.</a> Genes Dev. 24 (18), 1967-2000 (2010).</li><li>Sequeiros, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+markers+for+prostate+cancer+in+formalin-fixed+paraffin-embedded+tissues.">Molecular markers for prostate cancer in formalin-fixed paraffin-embedded tissues.</a> Biomed Res Int. 2013, 283635 (2013).</li><li>Cary, K. C., Cooperberg, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+in+prostate+cancer+surveillance+and+screening:+past,+present,+and+future.">Biomarkers in prostate cancer surveillance and screening: past, present, and future.</a> Ther Adv Urol. 5 (6), 318-329 (2013).</li><li>Sartori, D. A., Chan, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+in+prostate+cancer:+what's+new.">Biomarkers in prostate cancer: what's new.</a> Curr Opin Oncol. 26 (3), 259-264 (2014).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19 (1), 92-105 (2009).</li><li>Rodriguez, A., Griffiths-Jones, S., Ashurst, J. L., Bradley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mammalian+microRNA+host+genes+and+transcription+units.">Identification of mammalian microRNA host genes and transcription units.</a> Genome Res. 14 (10A), 1902-1910 (2004).</li><li>Borchert, G. M., Lanier, W., Davidson, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+III+transcribes+human+microRNAs.">RNA polymerase III transcribes human microRNAs.</a> Nat Struct Mol Biol. 13 (12), 1097-1101 (2006).</li><li>Cai, X., Hagedorn, C. H., Cullen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microRNAs+are+processed+from+capped,+polyadenylated+transcripts+that+can+also+function+as+mRNAs.">Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs.</a> RNA. 10 (12), 1957-1966 (2004).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+genes+are+transcribed+by+RNA+polymerase+II.">MicroRNA genes are transcribed by RNA polymerase II.</a> EMBO J. 23 (20), 4051-4060 (2004).</li><li>Bartel, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+target+recognition+and+regulatory+functions.">MicroRNAs: target recognition and regulatory functions.</a> Cell. 136 (2), 215-233 (2009).</li><li>Gordanpour, A., Nam, R. K., Sugar, L., Seth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+prostate+cancer:+from+biomarkers+to+molecularly-based+therapeutics.">MicroRNAs in prostate cancer: from biomarkers to molecularly-based therapeutics.</a> Prostate Cancer Prostatic Dis. 15 (4), 314-319 (2012).</li><li>Hurst, D. R., Edmonds, M. D., Welch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastamir:+the+field+of+metastasis-regulatory+microRNA+is+spreading.">Metastamir: the field of metastasis-regulatory microRNA is spreading.</a> Cancer Res. 69 (19), 7495-7498 (2009).</li><li>Saini, S., Majid, S., Dahiya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet,+microRNAs+and+prostate+cancer.">Diet, microRNAs and prostate cancer.</a> Pharm Res. 27 (6), 1014-1026 (2010).</li><li>Saini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+Role+of+mir-203+in+Prostate+Cancer+Progression+and+Metastasis.">Regulatory Role of mir-203 in Prostate Cancer Progression and Metastasis.</a> Clin Cancer Res. 17 (16), 5287-5298 (2011).</li><li>Ambs, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+profiling+of+microRNA+and+messenger+RNA+reveals+deregulated+microRNA+expression+in+prostate+cancer.">Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer.</a> Cancer Res. 68 (15), 6162-6170 (2008).</li><li>Martens-Uzunova, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+and+prognostic+signatures+from+the+small+non-coding+RNA+transcriptome+in+prostate+cancer.">Diagnostic and prognostic signatures from the small non-coding RNA transcriptome in prostate cancer.</a> Oncogene. 31 (8), 978-991 (2012).</li><li>Porkka, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiling+in+prostate+cancer.">MicroRNA expression profiling in prostate cancer.</a> Cancer Res. 67 (13), 6130-6135 (2007).</li><li>Schaefer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+and+prognostic+implications+of+microRNA+profiling+in+prostate+carcinoma.">Diagnostic and prognostic implications of microRNA profiling in prostate carcinoma.</a> Int J Cancer. 126 (5), 1166-1176 (2010).</li><li>Maugeri-Sacca, M., Coppola, V., De Maria, R., Bonci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+microRNAs+in+prostate+cancer+and+therapeutic+opportunities.">Functional role of microRNAs in prostate cancer and therapeutic opportunities.</a> Crit Rev Oncog. 18 (4), 303-315 (2013).</li><li>Xi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+microRNA+expression+of+RNA+extracted+from+fresh+frozen+and+formalin-fixed+paraffin-embedded+samples.">Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples.</a> RNA. 13 (10), 1668-1674 (2007).</li><li>Lucas, S. M., Heath, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+challenges+in+development+of+differentially+expressed+and+prognostic+prostate+cancer+biomarkers.">Current challenges in development of differentially expressed and prognostic prostate cancer biomarkers.</a> Prostate Cancer. , 640968 (2012).</li><li>Singh, S. K., Kumar, R., Wengel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+Novel+Bicyclo[2.2.1]+Ribonucleosides:+2'-Amino-+and+2'-Thio-LNA+Monomeric+Nucleosides.">Synthesis of Novel Bicyclo[2.2.1] Ribonucleosides: 2'-Amino- and 2'-Thio-LNA Monomeric Nucleosides.</a> J Org Chem. 63 (18), 6078-6079 (1998).</li><li>Suh, S. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-145+is+regulated+by+DNA+methylation+and+p53+gene+mutation+in+prostate+cancer.">MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer.</a> Carcinogenesis. 32 (5), 772-778 (2011).</li><li>Shukla, C. J., Pennington, C. J., Riddick, A. C., Sethia, K. K., Ball, R. Y., Edwards, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-capture+microdissection+in+prostate+cancer+research:+establishment+and+validation+of+a+powerful+tool+for+the+assessment+of+tumour-stroma+interactions.">Laser-capture microdissection in prostate cancer research: establishment and validation of a powerful tool for the assessment of tumour-stroma interactions.</a> BJU Int. 101 (6), 765-774 (2008).</li><li>Nelson, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAKE+and+LNA-ISH+reveal+microRNA+expression+and+localization+in+archival+human+brain.">RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain.</a> RNA. 12 (2), 187-191 (2006).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantification+of+microRNAs+by+stem-loop+RT-PCR.">Real-time quantification of microRNAs by stem-loop RT-PCR.</a> Nucleic Acids Res. 33 (20), e179 (2005).</li><li>Hanna, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+microRNAs+in+tissue+microarrays+by+in+situ+hybridization.">Quantitative analysis of microRNAs in tissue microarrays by in situ hybridization.</a> Biotechniques. 52 (4), 235-245 (2012).</li></ol>

The authors have no financial disclosures.

In this article, we describe a simplified workflow for miRNA expression profiling in archived FFPE or fresh frozen prostate cancer clinical tissues. In prostate cancer, several studies suggest an important role of microRNAs in prostate cancer initiation, progression and metastasis. However, conflicting results are often obtained on a specific miRNA22 since the miRNA extraction and analyses methods differ widely. In view of the emerging evidence supporting the potential application of miRNAs as alternative pros...

Cancer of the prostate gland is a commonly diagnosed male malignancy that is one of the leading causes of cancer related mortality among men. In US, an estimated 220,800 new cases and 27,540 deaths will be reported in 20151.
Prostate cancer is a heterogeneous disease with highly variable disease course- tumors can be indolent or very aggressive. A critical challenge in prostate cancer clinical management is posed by the inadequacy of currently used methods/biomarkers for disease screening, diagnosis, prognosis and treatment2. Current screening methods include prostate specific antigen (PSA) testing and a digital rectal examination (DRE) followed by prostate biopsies3. Prostate specific antigen (PSA) is the most widely used prostate cancer biomarker that has significantly revolutionized clinical management and improved survival rates4. However, due to inherent limitations of PSA including lack of specificity, PSA-based screening has led to over diagnosis and over treatment of the disease. In view of this, intensive efforts are being directed towards a search for alternate prostate cancer biomarkers, particularly those which can predict the aggressiveness of the disease and drive better treatment decisions4,5. Over the last few years, microRNAs (miRNAs) have emerged as promising alternate prostate cancer biomarkers.
MicroRNAs (miRNAs) constitute an evolutionarily conserved class of small non-coding RNAs that suppress gene expression post-transcriptionally via sequence-specific interactions with the 3&#8217;- untranslated regions (UTRs) of cognate mRNA targets. It is estimated that &#62;60% of mRNAs are conserved targets of miRNAs6. miRNA genes are located in intergenic regions or within introns or exons of protein/non-protein coding genes7. These genes are preferentially transcribed by RNA Polymerase II into primary miRNAs (pri-miRNAs, several kilobases long) that form hairpin shaped stem loop secondary structures. These pri-miRNAs are processed into precursor miRNAs (pre-miRNAs, 60-75 nucleotide long) that are exported to the cytoplasm and further processed into mature miRNAs (18-25 nucleotide long)8-10. miRNAs regulate key cellular processes including proliferation, development, differentiation and apoptosis11. Studies suggest a widespread dysregulation of miRNA expression profiles in various human malignancies including prostate cancer12-15. miRNA expression profiles have been reported to be widely dysregulated in primary and metastatic prostate cancer. Altered miRNA expression have been linked with prostate cancer progression, aggressiveness and recurrence highlighting the prognostic potential of miRNAs12,14,16-19. A growing body of evidence indicates that miRNAs play important mechanistic roles in prostate cancer initiation, development, progression and metastasis. Overall, miRNAs are emerging as promising alternate biomarkers for prostate cancer diagnosis and prognosis that can distinguish between normal and cancer tissues and aid in stratification of prostate tumors12. Also, miRNAs are important targets for development of effective therapeutics against prostate cancer20.
Owing to their small size and resistance to endogenous RNase activity, miRNAs are stable biomarkers that can be readily detected in formalin-fixed tissues21 and in prostate biopsies22. Moreover, the expression profiles of miRNAs have been compared in frozen and formalin fixed tissues and have been found to be strongly correlated21. However, miRNA expression profiling in prostate cancer clinical tissues is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. Here we describe a simplified workflow used in our lab for miRNA expression profiling in archived FFPE or fresh frozen prostate cancer clinical specimens. We employ a combination of quantitative real-time PCR and in situ hybridization for miRNA analyses of clinical specimens, with the former yielding more quantitative information and the latter for visualizing the differential expression of potential miRNA biomarkers in an array of tissues. Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues, expression analyses by Taqman-probe based miRNA RT-PCR and miRNA in situ hybridization technique using locked nucleic acid (LNA)-based probes23. LNA-based probes offer increased sensitivity and specificity compared to DNA- or RNA- based probes and enables robust detection of all miRNA sequences, regardless of their GC content and also allow discrimination of miRNA families. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA), with the latter offering the potential to accelerate miRNA biomarker discovery.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems&#160;</td>
			<td>RM2255</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus Autopix for LCM</td>
			<td>Arcturus/ Life Technologies</td>
			<td>LCM1621/LCM1110</td>
			<td>Alternatively, Arcutus Xt system from Life Technolgies can be used.&#160;</td>
		</tr>
		<tr>
			<td>CapSure Macro LCM Caps</td>
			<td>Life Technologies</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy FFPE Kit&#160;</td>
			<td>Qiagen</td>
			<td align="right">217504</td>
			<td></td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4351106</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman MicroRNA Reverse Transcription kit&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4366596</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman Fast Universal PCR master mix&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG labeled LNA probe for U6</td>
			<td>Exiqon</td>
			<td>99002-01</td>
			<td></td>
		</tr>
		<tr>
			<td>BM Purple AP substrate</td>
			<td>Roche</td>
			<td align="right">11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-1</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-2</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-8</a></td>
			<td></td>
		</tr>
		<tr>
			<td>AP-conjugated anti-digoxigenin antibody</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-7</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting media&#160;</td>
			<td>Vector Laboratories&#160;</td>
			<td>H-5501&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (guanidine isothiocyanate-phenol reagent)&#160;</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/15596018">15596-018</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Harris hematoxylin</td>
			<td>Statlab</td>
			<td>SL200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Statlab</td>
			<td>SL201&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

mirna expression analyses in prostate cancer clinical tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Expression profiling of miR-203 in LCM primary prostate cancer clinical specimens by RT-PCR analyses (Figure 1)
RT-PCR analyses of relative miR-203 expression in LCM primary prostate cancer tissues and the matched adjacent normal regions was carried out as described in Saini et al.15 RNU48 was used as a control. The Table below summarizes the relative miR-203 expression in prostate cancer tumor tissues relative to adjacent normal tissues.
<p class="jove...

Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

mirna-expression-analyses-in-prostate-cancer-clinical-tissues

Research

JoVE Journal

Medicine

10.8K visualizações.  University of California San Francisco. Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).