This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1. Prepare siRNA solutions
<ol>
	<li>Resuspend 5 nmol of scrambled (Ctrl) and 5 nmol of BRCA2 siRNA dried pellets in 100 &#181;l of RNase-free water (final siRNA concentration: 50 &#181;M). This is the siRNA stock and must be stored at -20 &#176;C in 10 &#181;l aliquots.</li>
	<li>Take an aliquot of 10 &#181;l from the siRNA stock and add 40 &#181;l of RNase-free water to obtain the 10 &#181;M siRNA working solution. This dilution must be stored at -20 &#176;C until use. 
	NOTE: The siRNA working solution...

<ol>
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M., 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=Germline+mutations+in+BRCA1+and+BRCA2+in+breast-ovarian+families+from+a+breast+cancer+risk+evaluation+clinic.">Germline mutations in BRCA1 and BRCA2 in breast-ovarian families from a breast cancer risk evaluation clinic.</a> J Clin Oncol. 19, 2247-2253 (2001).</li><li>Castro, E., Eeles, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+BRCA1+and+BRCA2+in+prostate+cancer.">The role of BRCA1 and BRCA2 in prostate cancer.</a> Asian J Androl. 14, 409-414 (2012).</li><li>Vencken, P. 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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=Mitochondrial+DNA+depletion+sensitizes+cancer+cells+to+PARP+inhibitors+by+translational+and+post-translational+repression+of+BRCA2.">Mitochondrial DNA depletion sensitizes cancer cells to PARP inhibitors by translational and post-translational repression of BRCA2.</a> Oncogenesis. 2, e82 (2013).</li><li>Yang, F., Guo, X., Yang, G., Rosen, D. G., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AURKA+and+BRCA2+expression+highly+correlate+with+prognosis+of+endometrioid+ovarian+carcinoma.">AURKA and BRCA2 expression highly correlate with prognosis of endometrioid ovarian carcinoma.</a> Mod Pathol. 24, 836-845 (2011).</li><li>Guaragnella, N., Marra, E., Galli, A., Moro, L., Giannattasio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+BRCA2+decreases+anoikis+and+its+heterologous+expression+sensitizes+yeast+cells+to+acetic+acid-induced+programmed+cell+death.">Silencing of BRCA2 decreases anoikis and its heterologous expression sensitizes yeast cells to acetic acid-induced programmed cell death.</a> Apoptosis. 19, 1330-1341 (2014).</li><li>Mahato, R. I., Rolland, A., Tomlinson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid-based+gene+delivery+systems:+pharmaceutical+perspectives.">Cationic lipid-based gene delivery systems: pharmaceutical perspectives.</a> Pharm Res. 14, 853-859 (1997).</li><li>Su, L. K., 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=Characterization+of+BRCA2:+temperature+sensitivity+of+detection+and+cell-cycle+regulated+expression.">Characterization of BRCA2: temperature sensitivity of detection and cell-cycle regulated expression.</a> Oncogene. 17, 2377-2381 (1998).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76, 4350-4354 (1979).</li><li>Burnette, W. N. '. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Western+blotting':+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Anal Biochem. 112, 195-203 (1981).</li><li>Hunter, F. 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E., 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=Specific+killing+of+BRCA2-deficient+tumours+with+inhibitors+of+poly(ADP-ribose)+polymerase.">Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.</a> Nature. 434, 913-917 (2005).</li><li>Ihnen, M., 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=Therapeutic+potential+of+the+poly(ADP-ribose)+polymerase+inhibitor+rucaparib+for+the+treatment+of+sporadic+human+ovarian+cancer.">Therapeutic potential of the poly(ADP-ribose) polymerase inhibitor rucaparib for the treatment of sporadic human ovarian cancer.</a> Mol Cancer Ther. 12, 1002-1015 (2013).</li><li>Jeong, J. H., Jo, A., Park, P., Lee, H., Lee, H. 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The authors have nothing to disclose.

Because germline mutations of the BRCA2 gene lead to increased risk of several cancer types, including female and male breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and melanoma 3,4, a number of studies have been undertaken to understand the biological function of the BRCA2 protein. Most of these studies are genetic-based mainly due to technical difficulties in analyzing a giant protein like BRCA2. The method described here for silencing and analyzing BRCA2 protein expression prov...

The BRCA2 (BReast CAncer susceptibility gene-2) gene encodes for a tumor suppressor protein that plays a crucial role in repairing DNA double-strand breaks by regulating the function of the recombinase enzyme Rad51 1. BRCA2 has also been implicated in the modulation of transcription and in cell cycle control 2. Germline mutations in the BRCA2 gene induce an autosomal dominant susceptibility to breast and ovarian cancer in women and prostate cancer in men, as well as predisposition to other cancer types 3,4. However, despite the increased risk in developing cancer, BRCA2 mutations that suppress or reduce BRCA2 function may render cancer cells more vulnerable to chemotherapeutic agents that cause DNA damage 5-7. Sporadic cancers exhibit a low rate of BRCA2 mutations (&#60; 3%) though reduced levels of BRCA2 protein have been detected in some cancer types, suggesting that BRCA2 protein may be lost during tumorigenesis in sporadic cancers through non-mutational-dependent mechanisms 7,8. Thus, it is important to fully understand BRCA2 functions in the context of cancer biology as well as in other biological settings.
A powerful tool used to identify novel functions of a gene in mammalian cells is to silence its expression. As an example, silencing BRCA2 expression in a variety of normal epithelial cells has recently led to the identification of a novel function of BRCA2 as regulator of anoikis resistance, an important step during acquisition of cancer cell invasive and metastatic ability 9. Gene silencing can be achieved by introducing in the cells either small interfering (si) RNA or short hairpin (sh) RNA molecules targeting the specific gene. The high potency of siRNA and its ease of use make it the preferential tool for silencing experiments aimed at gaining new insights into critical biological processes and to identify novel therapeutic targets. However, efficient knockdown of gene expression may not be easily achievable for all genes and may be highly variable depending on the cell type. Because a decrease in intracellular protein levels is the most relevant phenotype under investigation, it is crucial to quantify gene silencing by immunoblotting analysis of the gene product. With this respect, the BRCA2 protein presents a further challenge: being a large protein (approximately 390 kDa), technical difficulties do exist for conventional biochemical analysis, including immunoblotting.
We report here a protocol optimized for efficient silencing of BRCA2 in human epithelial cell lines and for rapid and successful detection of BRCA2 protein knockdown by immunoblotting analysis.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BRCA2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-003462-00</td>
			<td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td>
		</tr>
		<tr>
			<td>Ctrl siRNA</td>
			<td>Dharmacon</td>
			<td>&#160;D-001810-10-05</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Reagent&#160;</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/13778075">13778075</a></td>
			<td>Transfection Reagent</td>
		</tr>
		<tr>
			<td>OPTI-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Medium for transfection procedure</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td>
			<td>Life Technologies</td>
			<td>EA0378</td>
			<td>Gel for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample buffer&#160;</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing agent (10x)</td>
			<td>Life Technologies</td>
			<td>NP0004</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>Life Technologies</td>
			<td>NP0005</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>HiMark Pre-stained protein standard</td>
			<td>Life Technologies</td>
			<td>LC5699</td>
			<td>Prestained marker for gel electrophoresis</td>
		</tr>
		<tr>
			<td>XCell SureLoc Mini-Cell&#160; System</td>
			<td>Life Technologies</td>
			<td>EI0002</td>
			<td>Equipment for protein electrophoresis/transfer</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate SDS Running Buffer</td>
			<td>Life Technologies</td>
			<td>LA0041</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Life Technologies</td>
			<td>NP0006-1</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>BRCA2 H-300 rabbit polyclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8326</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Beta-tubulin monoclonal antibody</td>
			<td>Sigma Aldrich</td>
			<td>T4026-100UL</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Skim Milk powder</td>
			<td>Sigma Aldrich</td>
			<td>70166</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td>
			<td>Pierce Thermo Scientific</td>
			<td>34080/34096</td>
			<td>Chemiluminescence system</td>
		</tr>
		<tr>
			<td>PNT1A cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>95012614</td>
			<td>PNT1A human, normal prostate epithelium immortalized with SV40</td>
		</tr>
		<tr>
			<td>Nthy cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>90011609</td>
			<td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit I (MTT)</td>
			<td>Roche</td>
			<td>11465007001</td>
			<td>Kit for cell proliferation assays</td>
		</tr>
	</tbody>
</table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Before proceeding with a biological/biochemical assay to examine the effect of BRCA2 silencing on cell functions, the first step is to confirm the specificity of the silencing by using a scrambled siRNA (non-targeting siRNA) side by side with BRCA2 siRNA (Figure 1A). It is important to perform a second round of siRNA transfection with a high siRNA/transfection ratio to get a high efficiency of BRCA2 silencing. Indeed, performing only one round of siRNA transfection or using a lower siRN...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

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9.0K visualizações.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 