The work was supported by (1) a Breast Cancer Project Scholarship (2014-2016) funded by the Action for Breast Cancer Foundation and ALIVE 2013 through the Research, Innovation &#38; Development Trust (RIDT) of the University of Malta, (2) the Faculty of Medicine &#38; Surgery, University of Malta and (3) Project ACT financed by the Malta Council for Science &#38; Technology through FUSION: The R&#38;I Technology Development Programme 2016. The publication of this manuscript is supported through the Jove-Luminex grant.

Ethical approval for use of breast tissue material in this study was obtained from the University Research Ethics Committee (UREC) of the University of Malta (Ref: 22/2012).
1. Tissue Preparation
<ol>
	<li>Using appropriate H&#38;E procedures14, prepare stained reference tissue sections and digitally scan the slides for reference during microdissection.</li>
	<li>Using a microtome, cut 20 &#181;m tissues sections onto an RNAse-free water bath at 40 &#176;C. Collect the sections on membrane slides for laser microdissection using clean, RNase-decontaminated equipment and materials.</li>
	<li>Dry the membrane slides at 37 &#176;C in a drying incubator overnight. Stain using the appropriate H&#38;E procedure15 with molecular biology grade solutions and increasing the staining time of Hematoxylin to 6 min. If required, stain the membrane slides with the appropriate immunohistochemical protocols16.</li>
</ol>
2. Laser Microdissection
<ol>
	<li>Set the slide limits and calibrate the stage movement.
	<ol>
		<li>Move the laser view onto an empty area of a membrane slide. Calibrate the laser focus by firing a laser shot at increasing focus intervals (5%) until the laser starts to pierce through the membrane. Draw and microdissect any shape and finetune the laser focus to maximize the laser efficiency during the laser dissection.</li>
		<li>Increase the stage movement speed to a point where the laser dissection efficiency is not compromised. Calibrate the laser at the selected objective lens and re-calibrate if changing objectives (laser speed at 1&#8211;15%, focus at 70&#8211;75%, and power at 90&#8211;100% when using the 4X objective).</li>
	</ol>
	</li>
	<li>Scan the stained membrane slides using the 4X objective.</li>
	<li>Select and encircle the target areas for microdissection on the membrane slide. Laser dissect a minimum area of 42 mm2. Record the area dissected to determine the volume of sample homogenizing buffer to be used.</li>
	<li>Use the cap lift mechanism to retrieve the dissected section onto the diffuser caps of labeled tubes attached to the appropriate appendage. If the dissected section is not retrieved onto the cap, repeat the laser dissection of the area.</li>
</ol>
3. Tissue Lysis
Note: Several solutions and materials are supplied along with kits mentioned in the Table of Materials.
<ol>
	<li>Prepare the required homogenizing mixture volume for sample lysis using homogenizing solution and proteinase K at a 60:1 ratio.</li>
	<li>Pipette 2.4 &#181;L of homogenizing solution into each tube for each 1 mm2 of tissue area, vortex for 10 s at the maximum speed, and centrifuge at room temperature for 5 s at 2,500 x g.</li>
	<li>Incubate in a heating block at 65 &#176;C for 12&#8211;18 h with shaking at 600 rpm.</li>
	<li>Centrifuge the lysates at 21,000 x g for 5 min at room temperature.</li>
	<li>Using a pipette, carefully aspirate the clear supernatant and dispense into a labeled fresh tube. Avoid aspirating the tissue/membrane fragments as this may reduce the quality of results.</li>
	<li>Store the clear supernatant in a -80 &#176;C freezer.</li>
</ol>
4. Hybridization-based Assay
<ol>
	<li>Perform the following preparations.
	<ol>
		<li>Pre-warm the lysis mixture by placing the reagent bottle in an incubator at 37 &#176;C for 30 min and mix gently by inversion.</li>
		<li>If the tissue homogenates are frozen, thaw at room temperature and incubate at 37 &#176;C for 30 min. Vortex the samples at the maximum speed following the incubation.</li>
		<li>Set the shaking incubator at 54 &#176;C and place the probe of the temperature validation kit in an empty well of a 96-well plate. After 2 h check the temperature on the digital thermometer and set the delta temperature of shaking incubator to correct for any temperature difference.</li>
		<li>Thaw and vortex the probe set and blocking reagent for 10 s, centrifuge at room temperature for 5 s at 2,500 x g, and keep on ice.</li>
		<li>Keep the Proteinase K vial on ice during use.</li>
		<li>Sonicate the capture beads at 46 KHz for 3 min, then vortex at the maximum speed for another 3 min.</li>
	</ol>
	</li>
	<li>Prepare a 25 &#181;L working bead mix per well by scaling up the following reagents accordingly: 4.25 &#181;L of Nuclease-free water, 16.65 &#181;L of lysis mixture, 1.00 &#181;L of blocking reagent, 0.10 &#181;L of Proteinase K, 0.50 &#181;L of capture beads, 2.50 &#181;L of probe set.</li>
	<li>Vortex the working bead mix for 10 s at maximum speed, then pipette 25 &#181;L/well into the magnetic separation plate.</li>
	<li>Pipette 25 &#181;L tissue homogenate to a magnetic separation 96-well plate and 25 &#181;L homogenizing solution to 3 wells as the blanks.</li>
	<li>Seal the magnetic separation plate using a clear plastic pressure seal and place the plate in the incubator at 54 &#177; 1 &#176;C for 18&#8211;22 h while shaking at 600 rpm.</li>
	<li>Prepare a 1X wash buffer solution for 24 wells by mixing 31.5 mL of RNase-free water with 0.1 mL of wash buffer component 1 and 1.67 mL of wash buffer component 2.</li>
	<li>Remove the plate from incubator. Set the temperature of the shaking incubator to 50 &#177; 1 &#176;C.</li>
	<li>Mount the magnetic separation plate on the hand-held magnetic plate washer and lock in place. Remove the pressure seal and wait 1 min for the magnetic beads to settle.</li>
	<li>Washing Step: Invert the magnetic separation plate mounted onto the plate over the sink and blot gently on a tissue paper to remove residual solution. Using a multichannel pipette add 100 &#181;L of 1X wash buffer solution into each well and wait 15 s. Repeat this step to wash the plate 3 times.</li>
	<li>Pipette 50 &#181;L of the pre-amplifier reagent to each well. Seal the plate with an aluminum plate seal and shake the plate at 800 rpm for 1 min at room temperature. Incubate for 1 h at 50 &#177; 1 &#176;C while shaking at 600 rpm.</li>
	<li>Repeat step 4.9 (Washing step).</li>
	<li>Pipette 50 &#181;L of the amplifier reagent to each well. Seal the plate with an aluminum plate seal and shake the plate as in step 4.10.</li>
	<li>Repeat step 4.9 (Washing step).</li>
	<li>Vortex the label probe for 10 s at maximum speed. Add 50 &#181;L of the label probe to each well. Seal and shake the plate (step 4.10).</li>
	<li>Repeat Step 4.9 (Washing step).</li>
	<li>Vortex the Streptavidin phycoerythrin (SAPE) for 10 s at maximum speed. Add 50 &#181;L of SAPE to each well. Seal and shake the plate (step 4.10).</li>
	<li>Repeat Step 4.9 (Washing step) except use the SAPE wash buffer instead of the wash buffer solution.</li>
	<li>Add 130 &#181;L of SAPE wash buffer to each well and seal plate with an aluminum plate seal. Shake the plate at 800 rpm at room temperature for 3 min.</li>
	<li>Start-up the magnetic bead analyzer. Perform the calibration and verification routine by following the manufacturer&#8217;s instructions.</li>
	<li>Set up a protocol on the magnetic bead analyzer using the following parameters: Sample Size: 100 &#181;L; DD Gate: 5,000&#8211;25,000; Timeout: 90 s; Bead Event/Bead Region: 100; and click next. Select the respective bead numbers in the panel and define the bead regions with the respective target genes as indicated by the manufacturer.</li>
	<li>Add a new analysis by selecting &#8220;Create Batch using an existing protocol&#8221; from the Batches tab. In the plate layout designate the wells as unknown or blank, respectively and provide a label for each sample in the Samples Panel.</li>
	<li>Remove the aluminum plate seal from the 96-well reaction plate. Eject the plate tray by clicking on the Eject button, load the plate into the magnetic bead analyzer. Click Retract| Run Batch to initiate the analyzer. After reading, eject and remove the 96-well plate. Clean and shut down the magnetic bead analyzer as recommended by the manufacturer.</li>
</ol>
5. Data Analysis
<ol>
	<li>In the Batch| Results tab, select the respective analysis file and click the export to.csv button. Open the.csv file using a spreadsheet software.</li>
	<li>Observe the bead counts and omit any results with &#60; 40 beads/region.</li>
	<li>Average out the values of the blank wells and work out the standard deviation (SD) and Limit of Detection (LOD) (blank average + (3xSD)) per target gene. Set the values lower than the LOD as undetected. 
	Note: If any of the normalizing expression values are undetected, regard the sample data as inadequate. Consider expression values over 20,000 as over the upper sensitivity limit. The lysates of these samples should be diluted and re-analyzed.</li>
	<li>Subtract the blank average from the raw data per gene.</li>
	<li>Compute the geometric mean of the selected normalizing genes per sample. Normalize individual sample data to the respective geometric mean.</li>
	<li>Analyze the data on data science platforms or using defined algorithms.
	<ol>
		<li>Import the data into the data science platform by clicking Add Data. Drag the data file into the Process Workspace and connect to a [Select Attributes] operator, then to a Principal component analysis (PCA) operator, and then to the result port.</li>
		<li>In the Select Attribute operator select the subset of normalized gene data and a nominal variable such as breast cancer subtype. Run the process by clicking Play. In the Charts Tab select the &#8220;Scatter 3D Color&#8221; in the Chart style and the nominal variable in the Color field. Assess the PCA data variability on a 3-dimensional plot.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Abrahamsen, H. N., Steiniche, T., Nexo, E., Hamilton-Dutoit, S. J., Sorensen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+quantitative+mRNA+analysis+in+paraffin-embedded+tissues+using+real-time+reverse+transcriptase-polymerase+chain+reaction:+a+methodological+study+on+lymph+nodes+from+melanoma+patients.">Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients.</a> J Mol Diagn. 5 (1), 34-41 (2003).</li><li>Macabeo-Ong, 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=Effect+of+duration+of+fixation+on+quantitative+reverse+transcription+polymerase+chain+reaction+analyses.">Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses.</a> Mod Pathol. 15 (9), 979-987 (2002).</li><li>Yang, W., 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=Direct+quantification+of+gene+expression+in+homogenates+of+formalin-fixed,+paraffin-embedded+tissues.">Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.</a> Biotechniques. 40 (4), 481-486 (2006).</li><li>Flagella, 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=A+multiplex+branched+DNA+assay+for+parallel+quantitative+gene+expression+profiling.">A multiplex branched DNA assay for parallel quantitative gene expression profiling.</a> Anal Biochem. 352 (1), 50-60 (2006).</li><li>Goldhirsch, 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=Strategies+for+subtypes--dealing+with+the+diversity+of+breast+cancer:+highlights+of+the+St.+Gallen+International+Expert+Consensus+on+the+Primary+Therapy+of+Early+Breast+Cancer+2011.">Strategies for subtypes--dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011.</a> Ann Oncol. 22 (8), 1736-1747 (2011).</li><li>Schnitt, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+and+prognosis+of+invasive+breast+cancer:+from+morphology+to+molecular+taxonomy.">Classification and prognosis of invasive breast cancer: from morphology to molecular taxonomy.</a> Mod Pathol. 23, S60-S64 (2010).</li><li>Bae, Y. 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=Fibronectin+expression+in+carcinoma+cells+correlates+with+tumor+aggressiveness+and+poor+clinical+outcome+in+patients+with+invasive+breast+cancer.">Fibronectin expression in carcinoma cells correlates with tumor aggressiveness and poor clinical outcome in patients with invasive breast cancer.</a> Hum Pathol. 44 (10), 2028-2037 (2013).</li><li>Park, J., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+epithelial+cell+interactions+with+fibronectin+stimulate+epithelial-mesenchymal+transition.">Mammary epithelial cell interactions with fibronectin stimulate epithelial-mesenchymal transition.</a> Oncogene. 33 (13), 1649-1657 (2014).</li><li>Zhou, Z., 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=MRI+detection+of+breast+cancer+micrometastases+with+a+fibronectin-targeting+contrast+agent.">MRI detection of breast cancer micrometastases with a fibronectin-targeting contrast agent.</a> Nat Commun. 6, 7984 (2015).</li><li>Antonyak, M. 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=Cancer+cell-derived+microvesicles+induce+transformation+by+transferring+tissue+transglutaminase+and+fibronectin+to+recipient+cells.">Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells.</a> Proc Natl Acad Sci U S A. 108 (12), 4852-4857 (2011).</li><li>Moon, P. G., 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=Fibronectin+on+circulating+extracellular+vesicles+as+a+liquid+biopsy+to+detect+breast+cancer.">Fibronectin on circulating extracellular vesicles as a liquid biopsy to detect breast cancer.</a> Oncotarget. 7 (26), 40189-40199 (2016).</li><li>Parker, J. 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=Supervised+risk+predictor+of+breast+cancer+based+on+intrinsic+subtypes.">Supervised risk predictor of breast cancer based on intrinsic subtypes.</a> J Clin Oncol. 27, (2009).</li><li>Nielsen, 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=Analytical+validation+of+the+PAM50-based+Prosigna+Breast+Cancer+Prognostic+Gene+Signature+Assay+and+nCounter+Analysis+System+using+formalin-fixed+paraffin-embedded+breast+tumor+specimens.">Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens.</a> BMC Cancer. 14, 177-177 (2014).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+Eosin+Staining+of+Tissue+and+Cell+Sections.">Hematoxylin and Eosin Staining of Tissue and Cell Sections.</a> Cold Spring Harbor Protocols. (5), (2008).</li><li>Lin, F., Prichard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Practical Immunohistochemistry: Frequently asked questions. , (2011).</li><li>Grech, G., 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+Classification+of+Breast+Cancer+Patients+Using+Formalin-fixed+Paraffin-embedded+Derived+RNA+Samples.">Molecular Classification of Breast Cancer Patients Using Formalin-fixed Paraffin-embedded Derived RNA Samples.</a> Journal of Molecular Biomarkers &amp; Diagnosis. 7 (S8), (2016).</li></ol>

The Invitrogen&#8482; QuantiGene&#8482; Plex Assay&#160;is proprietary of Thermo&#160;Fisher Scientific.&#160;Production and Open Access publication of this video-article was sponsored by Luminex Corporation

A bead-based multiplex bDNA assay was optimized to quantify gene expression on degraded RNA derived from FFPE breast cancer tissue and normal breast ducts. Optimizing the assay, involved developing an algorithm to classify breast cancer tumors in luminal and basal subtypes utilizing 8 well-known biomarkers and 5 potential normalizing genes. Data normalization was done using permutations of the normalizing genes. The selection of the normalizing genes was based on the best prediction of receptor status using the Luminal/Basal classifier genes. To classify Luminal/Basal subtypes from FFPE tissues, the normalizing genes selected were Beta-actin (ACTB), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Hypoxanthine Phosphoribosyltransferase 1 (HPRT1).
The method can be adapted for use in other diagnostic and research areas following adequate selection of the normalizing gene set. One important application of this method in the research sector is the measurement of biomarkers in archival material that is well annotated with clinical outcomes. This could validate potential predictive markers in retrospective studies, quickly and accurately, and avoid long-term prospective studies awaiting disease-free survival and overall survival data. Currently our group is investigating the use of the assay to detect receptor-positive exosomes, which requires the development of a new algorithm using alternate normalizing genes for data normalization. The use of liquid biopsies and robust gene expression assays will allow high throughput multiplex assays adapted for patient management during treatment, and provide a means to follow treatment efficacy, potential relapse due to resistance to therapy, and the metastatic capacity of the tumor.
This method also has a wide range of possible applications in the diagnosis of tumors and is adapted to the current diagnostic workflow. The main advantages of this method in the diagnostic field include: (1) implementation of high throughput assays, (2) excluding subjectivity and equivocal results originating from image-based measurements, (3) accurate detection of multiple targets simultaneously, which enhance accuracy and minimize the use of precious patient samples, and (4) no requirement for highly specialized facilities and human resources. The optimized sampling process, together with the low input of material required for the bead-based multiplex assay, allows further investigation of tumor heterogeneity; by using laser microdissection to accurately separate multiple foci of malignant tissue from the same patient section, it is possible to compare multiple gene expression between them as well as with matched normal tissue (Figure 4). Low material input is vital for diagnostic application on tumor biopsies that provide limited tumor tissue. The capacity of the assay to measure gene expression from degraded RNA samples allows easy transportation of samples for analysis within an institution or to servicing laboratories. In addition, whole section analysis was also possible using H&#38;E stained material (Figure 1).
For the success of this protocol, it is imperative to: (1) ensure proper sampling of the tumor site/s that are lysed for the assay and (2) develop well optimized and validated data normalization algorithms, for each gene expression panel and/or individual prognostic or predictive biomarkers. The former depends on the technical experience of the technician/scientist performing the sampling. It is recommended to take an additional core and prepare a tissue microarray (TMA) in the same format of the multiplex magnetic bead assay (96-well format). This will provide an archive of tumor sites as a replica of samples used for the RNA-based assay. TMAs can also be assessed with other techniques for follow-up research or validation of results. The development of normalization algorithms is dependent on the material being investigated and the normalizing genes selected for normalization. Different panels of normalizing genes are selected based on the level and variability of expression in the sample analyzed and this varies between cancer tissues from different origins, exosomes from plasma, or circulating tumor cells. Validation of the assay includes sample processing since various preparations will also result in different normalization algorithms.
To summarize, the use of bDNA technology in combination with magnetic bead technology and the selection of the proper panel of target genes, will provide the added advantage of measuring gene expression directly in tissue lysates derived from small amounts of patient material, including microdissected material, exosomes, and circulating tumor cells. In addition to detection of tumor heterogeneity, the proper use of panels has the potential to detect tumor derived exosomes for early diagnostics and early detection of relapses. Since there is no need for a nucleic acid amplification step, the signal amplification using the bDNA technology, combined with the bead-based multiplex, measures multiple gene expression in clinically-annotated archival material and provide a resource for biomarker validation.

Optimization of RNA based assays using archival formalin-fixed paraffin-embedded (FFPE) material is challenging due to variability in surgical tissue processing and degradation of RNA caused by formalin used for tissue integrity preservation1,2. To overcome the limitation of performing accurate gene expression studies from archival material, our group used the bDNA multiplex magnetic bead assay. Instead of enzymatic amplification of a target template, the bDNA technology uses hybridization of specific probes and amplification of a reporter signal3. The short recognition sequences of the capture and detection probes are designed to hybridize to short fragments of target RNA4. In addition, the use of tissue homogenates as the direct starting material in this assay, overcomes the inevitable loss of RNA that results from assays requiring prior RNA extraction and purification. Signal amplification, the use of short recognition sequences, and the exclusion of a purification step, contribute to the reduce in technical variation of the assay. The technology provides the possibility to multiplex the assay (up to 80 RNA targets) and measure the expression of a panel of targets from low material input. This protocol describes the preparation and staining of tissue samples for laser microdissection. Staining on the membrane slides facilitate the imaging of the tumor and histological architecture to provide accurate selection and profiling of: (1) the tumor and normal ducts in breast tissue, and (2) the malignant cell clones within heterogeneous tumors.
Molecular classification of breast cancer is a process that interrogates molecular markers to categorize patient tumors into three molecular classes, i.e., luminal, human epidermal growth factor receptor 2 (HER2)-enriched, and basal subtype. The HER2-enriched subtype is well defined, with high expression of HER2 receptor, due to the ERBB2 gene amplification, combined with low or absent estrogen receptor (ER) and progesterone receptor (PgR). The luminal subtype is generally positive for ER and the basal subtype are in general negative for the three receptors (HER2, ER, PgR), and significantly overlaps with the triple negative breast cancer (TNBC) diagnostic subtype5,6. Other markers are used to determine epithelial and mesenchymal characteristics. Fibronectin (FN1) is a main component of the breast tissue mesenchymal compartment. Increased FN1 expression is accompanied by high Ki67 staining, and shows a signature for a more invasive tumor7,8 and is associated with metastasis9. Interestingly, FN1 was found to be present in microvesicles originating from the tumor cells, that induced activation of mitogenic signals in recipient fibroblasts10. Hence, circulating microvesicles such as exosomes are a potential marker of early detection or metastasis and relapse11.
Tumor area selection for breast cancer transcriptional subtyping has recurrently been performed by macrodissection12,13. To overcome tissue heterogeneity and increase sensitivity, we have reliably combined classical tissue staining with multiplex molecular profiling methods. As a proof of principle, two distinct breast cancer clones have been defined by their epithelial mesenchymal signature and metastatic potential. The workflow of the described protocol can be easily translated to the current clinical setup and used to selectively isolate and characterize tissue subtypes using targeted mRNA profiling.

<table>
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Heamatoxylin Mayer&#39;s</td>
			<td>Sigma</td>
			<td>MHS16-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y Aquaeous solution</td>
			<td>Sigma</td>
			<td>HT110216-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Rabbit Serum</td>
			<td>Monosan</td>
			<td>MONX10963</td>
			<td>Working dilution: 1/40</td>
		</tr>
		<tr>
			<td>Biotinylated Rabbit anti-mouse</td>
			<td>Dako</td>
			<td>E0354</td>
			<td>Working dilution: 1/200</td>
		</tr>
		<tr>
			<td>ER antibody (6F11)</td>
			<td>Vector Laboratories</td>
			<td>VPE614</td>
			<td>Working dilution: 1/45</td>
		</tr>
		<tr>
			<td>HER2 antibody (CB11)</td>
			<td>Novocastra</td>
			<td>CB11-L-CE</td>
			<td>Working dilution: 1/325</td>
		</tr>
		<tr>
			<td>Ki67 antibody (MIB-1)</td>
			<td>Dako</td>
			<td>M7240</td>
			<td>Working dilution: 1/500</td>
		</tr>
		<tr>
			<td>Avidin Biotin Complex kit</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-E Inverted microscope</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td>4x, 10x, 20x and 40x objectives</td>
		</tr>
		<tr>
			<td>Laser Microdissection membranes</td>
			<td>Molecular Machines &#38;Industries</td>
			<td>S0103</td>
			<td></td>
		</tr>
		<tr>
			<td>mmi CellCamera 1.4</td>
			<td>Molecular Machines &#38;Industries</td>
			<td>MX4285c-ACK07</td>
			<td></td>
		</tr>
		<tr>
			<td>mmi Cellcut Plus</td>
			<td>Molecular Machines &#38;Industries</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diffuser caps</td>
			<td>Molecular Machines &#38;Industries</td>
			<td>50210</td>
			<td></td>
		</tr>
		<tr>
			<td>mmi Celltools Software v.4.01rcl</td>
			<td>Molecular Machines &#38;Industries</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Thermomixer comfort</td>
			<td>Eppendorf</td>
			<td>5355000038</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5mL heating block for Eppendorf Thermomixer</td>
			<td>Eppendorf</td>
			<td>22670522</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate heating block for Eppendorf Thermomixer</td>
			<td>Eppendorf</td>
			<td>22670565</td>
			<td></td>
		</tr>
		<tr>
			<td>Labnet Vortemp 56 Shaking incubator</td>
			<td>Labnet</td>
			<td>52056A-220</td>
			<td></td>
		</tr>
		<tr>
			<td>LX200 100/200</td>
			<td>Luminex</td>
			<td></td>
			<td>Magnetic bead analyser</td>
		</tr>
		<tr>
			<td>Invitrogen QuantiGene Sample Processing Kit - FFPE Tissues</td>
			<td>ThermoFisher Scientific</td>
			<td>QS0109</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen QuantiGene Plex 2.0 Assay Kit (Magnetic Separation)</td>
			<td>ThermoFisher Scientific</td>
			<td>QP1011</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermaseal RTS Sealing Film</td>
			<td>Thermaseal</td>
			<td>765246</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand-Held Magnetic Plate Washer</td>
			<td>ThermoFisher Scientific</td>
			<td>QP1011</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen QuantiGene Incubator Temperature Validation Kit</td>
			<td>Affymetrix/Panomics</td>
			<td>QS0517</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K (50&#181;g/&#181;L)</td>
			<td>ThermoFisher Scientific</td>
			<td>14622</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen QuantiGene Plex 2.0 Sets</td>
			<td>ThermoFisher Scientific</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi Speed Vortex</td>
			<td>Kisker Biotech</td>
			<td>MSV-3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Silvercrest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNASEZAP</td>
			<td>Sigma</td>
			<td>R2020-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium 96-well plate seal</td>
			<td>Sigma</td>
			<td>Z721549-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Validation Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>QS0517</td>
			<td></td>
		</tr>
		<tr>
			<td>RapidMiner Studio Community 7.1.001</td>
			<td>RapidMiner</td>
			<td></td>
			<td>Data Science Platform</td>
		</tr>
		<tr>
			<td>Hybridisation oven</td>
			<td>Hybaid (Thermo Scientific)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of a multiplex rna-based expression assay using breast cancer archival material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

The described method has been applied for the simultaneous measurement of 40 transcripts in H&#38;E stained (Figure 1), microdissected (Figure 2) highly degraded FFPE material. Using this method, we show the accurate characterization of receptor status (Figure 3A), classification of tumors into luminal and basal molecular subtypes17, and differential expression of the mesenchymal marker, FN1, when comparing tumor and matched control tissue (Figure 3B), in the various receptor positive and negative subtypes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57148/57148fig1.jpg" /> 
Figure 1: Gene expression using H&#38;E stained material. Correlation between an expression profile derived from an unstained tumor section as compared to a stained tumor section. [Pearson Correlation p-value = 5.34E-26]. Reproduced with permission17. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57148/57148fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57148/57148fig2.jpg" /> 
Figure 2: Laser microdissection of FFPE tissues. (A) H&#38;E stained slide; (B) Immunohistochemical staining for ER expression; (C) HER2 immunohistochemical staining; (D) Unstained 20 &#181;m section on laser microdissection membrane slides. The yellow arrow indicates an area of invasive tumor that is not clearly demarcated due to lack of staining. (E) A 20 &#181;m section stained with H&#38;E for better delineation of areas of interest. White arrows indicate laser dissection trail while the Red arrow shows the laser focus during dissection. All illustrations were captured at 10x magnification. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57148/57148fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57148/57148fig3.jpg" /> 
Figure 3: Expression of (A) receptor status and (B) mesenchymal marker FN1, in breast tumor compared to matched normal. The classical diagnostic breast cancer subtypes are defined as per diagnostic result using immunohistochemistry and fluorescence in situ hybridization (FISH) for HER2 equivocal immunohistochemical staining. The normalized expression of (A) HER2 and ER, (B) FN1 measured by the hybridization-based assay is illustrated in HER2 positive, ER positive, and TNBC cases compared to patient matched normal breast tissue. The Kruskal Wallis Test Statistic (K-W &#967;2) shows that the expression of HER2 is significantly (p &#60; 0.05) higher in the tumor tissue as opposed to the matched normal tissue in the HER2 positive cohort and significantly lower in the TNBC cohort. ER expression was not found to be significantly higher in the tumor tissue as opposed to the matched normal in the ER positive and TNBC cohorts. FN1 is significantly higher in the tumor tissue in the HER2 and ER positive subtypes and shows a trend towards being significantly elevated in tumor tissue also in the TNBC cohort. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57148/57148fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57148/57148fig4.jpg" /> 
Figure 4: Case study: tumor heterogeneity. Morphologically distinct tumors were microdissected and treated as distinct samples. (A) The master scan of the H&#38;E section. (B, C) A 10x and 40x magnification, respectively for each tumor morphology identified. (D) Immunohistochemical staining for ER at 10x magnification. (E) Immunohistochemical staining for Ki67 at 10x magnification showing a higher mitotic activity in tumor 1. (F) Normalized expression levels for the ESR1 gene in each tumor showing relatively high and equal expression between tumors as expected from the immunohistochemical result. (G) Normalized expression levels of FN1, a mesenchymal marker, where increased FN1 expression is accompanied by high Ki67 staining showing a signature for a more invasive tumor. The inverse is observed in tumor 2, which appears to be a slower proliferating tumor with a lower malignant potential represented by reduced FN1 expression. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57148/57148fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material at JoVE.com

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

optimization-multiplex-rna-based-expression-assay-using-breast-cancer

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.8K Views.  University of Malta. Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

Video: Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Summary

Explore More Videos

Chapters in this video

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High-throughput Protein Expression Generator Using a Microfluidic Platform

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay