The authors would like to thank the technical staff of the Translational Research Unit;&#160;Mary Economou, Jos&#233;&#160;Galv&#225;n, Caroline Hammer, Dominique M&#252;ller, Liliane Sch&#246;ni and the Informatics Team at the Institute of Pathology, University of Bern.

NOTE: This study has been approved by the local ethics committee of the Insel Hospital, Bern, Switzerland (07-10-13). Tissues were obtained from the Tumor bank Bern, Institute of Pathology, University of Bern.
1. Planning and Design (Consulting)
<ol>
	<li>Think about the research question to be answered. Decide on the tissue types to be included in the project. Determine what histological structures are important to answer the question.</li>
	<li>Ascertain the most useful core diameter and the number of cores per patient for the project. Decide on the number of copies of the next-generation Tissue Micro Array (ngTMA) based on the amount of biomarkers to be investigated.</li>
	<li>Consider statistical aspects such as sample size and analysis after the tissue microarray (TMA) is constructed. Determine the number of patients for inclusion in the study and establish control tissues for the array.</li>
	<li>Retrieve the histological (or diagnostic) Haematoxylin and Eosin (H&#38;E) slides of each patient. Briefly review the slides and identify the ones containing relevant information and histology for the project. 
	NOTE: Make new sections of the paraffin blocks, if special stain or immunohistochemistry slide is desired for slide scanning and annotation, instead of H&#38;E slides</li>
</ol>
2. Slide Scanning
<ol>
	<li>Turn on PC and slide scanner and open scanning software. Select &#8220;automatic mode&#8221; for bright field scanning from the preview screen.</li>
	<li>Click on &#8220;scan options&#8221; to adjust slide quality parameters. At this time, select whether slides are to be directly scanned to the digital platform accessible by web or to a local drive (or external drive) and set the types of scanning and number of focus points.</li>
	<li>Click on &#8220;Server Parameters&#8221; to scan to the &#8220;Case center&#8221;. At this step, add more magazines, label all slides and set the folder in Case Center where the slides should be stored. Each magazine holds up to 25 slides.</li>
	<li>Check quality/cleanliness of actual histological slides for scanning. If necessary, clean slides with 70% ethanol.</li>
	<li>Load up to 25 slides per magazine with the labels pointing inward (Figure 3A)</li>
	<li>Place the magazine in the scanner. For more than one magazine, place them on top of each other.</li>
	<li>Start scanning slide by clicking on the green arrow (Figure 3B). When all slides are scanned, unload the magazines and shut off the program, PC and slide scanner.</li>
</ol>
3. Digital Slide Annotation
<ol>
	<li>Enter the browser address for digital slide management center&#160;and download the free digital slide viewer software.</li>
	<li>Open the digital slide viewer software and select the folder containing the digital slides (Figure 4A).</li>
	<li>Add notes, reorder and manage the digital slide folder or assign user rights to colleagues or add attachments to the folder.</li>
	<li>Click on desired digital slide which appears in the digital slide viewer.</li>
	<li>Using the magnification tool, evaluate the slide and find the areas of interest for integration into the ngTMA.</li>
	<li>Using the &#8216;TMA annotation tool&#8217;, select the size of the desired core and the color of the annotation. Place the annotation on the digital slide by clicking on it (Figure 4B).</li>
	<li>Move the annotation to the desired histological structures for incorporation into the ngTMA (Figure 4C).</li>
	<li>Repeat this process of annotation again by using the annotation tool, selecting a core size and a desired annotation color. Repeat the slide viewing and annotation on all slides in the folder.</li>
	<li>Alternatively, place tissue cores for PCR into 0.2 ml tubes rather than integration into the TMA. Annotate slides using a different color to identify these spots for further molecular analysis.</li>
	<li>Create a list of all cases with their corresponding annotations and colors.</li>
</ol>
4. Tissue Microarray Construction
<ol>
	<li>Retrieve the corresponding paraffin tissue blocks for all annotated digital slides, Sort blocks in the desired order for tissue microarraying.</li>
	<li>Ensure that the tissue blocks have a minimum of 4 mm thickness. If not, reembedding of tissue is necessary.</li>
	<li>Turn the PC &#8220;ON&#8221; and automated tissue microarrayer instrument. Open the tissue microarrayer software and provide a name to the project</li>
	<li>Perform a &#8220;Tool Change&#8221; and select the required tool diameter for the project (i.e., 0.6, 1.0, 1.5, or 2.0 mm).</li>
	<li>Load up to 12 &#8216;recipient&#8217; TMA blocks into the machine. Give an ID to each of the 12 blocks</li>
	<li>Create a TMA layout for each recipient block. Observe a TMA design with number of rows, columns, and empty lines as well as distance between cores. Create a new layout for each recipient block or use the same layout repeatedly.</li>
	<li>Place the cursor on the recipient block layout to take the first core.</li>
	<li>Next, load up to 60 donor blocks into the tissue microarrayer (Figure 5A). Place ten blocks in each row A to F. Give each block a donor ID. Observe images of each donor block on the computer screen acquired automatically by the tissue microarrayer.</li>
	<li>Starting with the first block, click on &#8220;Slide&#8221;. Observe the annotated digital slide stored in the digital slide management center&#160;(or on the external drive) using the digital slide viewer. View the image of the donor block and the digital slide side-by-side.</li>
	<li>Select reference points on the block image to superimpose the donor block image with the digital slide</li>
	<li>After clicking &#8220;Next&#8221;, observe the annotations on a larger image of the block. (Figure 5B). Click on top of the annotations to confirm and click &#8220;Start&#8221;. 
	NOTE: This prompts the tissue microarrayer to start drilling a hole of 0.6 mm in diameter in the recipient block at the selected starting point. Then in a second step, using the punching tool, the instrument punches hole in the tissue from the selected donor block at the exact annotated and confirmed region. Cores are then transferred from the donor to the recipient block.</li>
	<li>After approximately 500 cores, clean the drill and punching tool using a swab of xylene.</li>
	<li>If tissue cores are desired (rather than punches into an ngTMA), click on the &#8220;PCR&#8221; tool for the block, confirm up to 4 annotations and hit &#8220;Start&#8221;. This will punch and transfer the cores into a 0.2 ml tube.</li>
	<li>Repeat point 4.9 to 4.11 with the second donor block, and so on.</li>
	<li>After using all the donor blocks, enter a second round of donor blocks (61-120) into the tissue microarrayer and continue steps 4.8 to 4.11.</li>
	<li>Refresh donor and recipient block images while the project progress while drilling and punching is occurring (Figure 5C).</li>
	<li>Perform an &#8220;Export&#8221; after the project is complete. Locate the .xslx file with the donor and recipient block IDs, the localization of all punches within the TMA as well as the TMA layout/design, in the exported folder. Save all donor block images with all superimposed annotation as jpg images in the exported folder (Figure 5D). The final TMA block is then ready.</li>
</ol>

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<li>Hsu, F. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+microarrays+are+an+effective+quality+assurance+tool+for+diagnostic+immunohistochemistry%5BTitle%5D)+AND+%22Mod+Pathol%22%5BJournal%5D)">Tissue microarrays are an effective quality assurance tool for diagnostic immunohistochemistry.</a> Mod Pathol. 15, 1374-1380 (2002).</li>
<li>Marin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+BRAF+p.V600E+Mutations+in+Melanoma+by+Immunohistochemistry+Has+a+Good+Interobserver+Reproducibility%5BTitle%5D)+AND+%22Arch+Pathol+Lab+Med%22%5BJournal%5D)">Detection of BRAF p.V600E Mutations in Melanoma by Immunohistochemistry Has a Good Interobserver Reproducibility.</a> Arch Pathol Lab Med. , 71-75 (2014).</li>
<li>Polley, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+International+Ki67+Reproducibility+Study%5BTitle%5D)+AND+%22J+Natl+Cancer+Inst%22%5BJournal%5D)">An International Ki67 Reproducibility Study.</a> J Natl Cancer Inst. , 1897-1906 (2013).</li>
<li>Zlobec, I., Terracciano, L., Jass, J. R., Lugli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Value+of+staining+intensity+in+the+interpretation+of+immunohistochemistry+for+tumor+markers+in+colorectal+cancer%5BTitle%5D)+AND+%22Virchows+Arch%22%5BJournal%5D)">Value of staining intensity in the interpretation of immunohistochemistry for tumor markers in colorectal cancer.</a> Virchows Arch. 451, 763-769 (2007).</li>
<li>Zlobec, I., Koelzer, V. H., Dawson, H., Perren, A., Lugli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+tissue+microarray+%28ngTMA%29+increases+the+quality+of+biomarker+studies%3A+an+example+using+CD3%2C+CD8%2C+and+CD45RO+in+the+tumor+microenvironment+of+six+different+solid+tumor+types%5BTitle%5D)+AND+%22J+Transl+Med%22%5BJournal%5D)">Next-generation tissue microarray (ngTMA) increases the quality of biomarker studies: an example using CD3, CD8, and CD45RO in the tumor microenvironment of six different solid tumor types.</a> J Transl Med. 11, 104(2013).</li>
<li>Yoshida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunohistochemical+detection+of+ROS1+is+useful+for+identifying+ROS1+rearrangements+in+lung+cancers%5BTitle%5D)+AND+%22Mod+Pathol%22%5BJournal%5D)">Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.</a> Mod Pathol. , 711-720 (2014).</li>
<li>Droeser, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+pattern+and+prognostic+significance+of+CD4%2B%2C+FOXP3%2B+and+IL-17%2B+tumor+infiltrating+lymphocytes+in+ductal+and+lobular+breast+cancers%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers.</a> BMC Cancer. 12, 134(2012).</li>
<li>Fleischmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Androgen+receptors+are+differentially+expressed+in+Gleason+patterns+of+prostate+cancer+and+down-regulated+in+matched+lymph+node+metastases%5BTitle%5D)+AND+%22Prostate%22%5BJournal%5D)">Androgen receptors are differentially expressed in Gleason patterns of prostate cancer and down-regulated in matched lymph node metastases.</a> Prostate. 71, 453-460 (2011).</li>
<li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pattern+of+breast+cancer+susceptibility+gene+1+expression+is+a+potential+prognostic+biomarker+in+resectable+pancreatic+ductal+adenocarcinoma%5BTitle%5D)+AND+%22Pancreas%22%5BJournal%5D)">Pattern of breast cancer susceptibility gene 1 expression is a potential prognostic biomarker in resectable pancreatic ductal adenocarcinoma.</a> Pancreas. 42, 977-982 (2013).</li>
<li>Fleischmann, A., Rotzer, D., Seiler, R., Studer, U. E., Thalmann, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Her2+amplification+is+significantly+more+frequent+in+lymph+node+metastases+from+urothelial+bladder+cancer+than+in+the+primary+tumours%5BTitle%5D)+AND+%22Eur+Urol%22%5BJournal%5D)">Her2 amplification is significantly more frequent in lymph node metastases from urothelial bladder cancer than in the primary tumours.</a> Eur Urol. 60, 350-357 (2011).</li>
<li>Zhang, Z. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+Annexin+A1+protein+expression+in+human+gastric+adenocarcinoma+using+proteomics+and+tissue+microarray%5BTitle%5D)+AND+%22World+J+Gastroenterol%22%5BJournal%5D)">Identification of Annexin A1 protein expression in human gastric adenocarcinoma using proteomics and tissue microarray.</a> World J Gastroenterol. 19, 7795-7803 (2013).</li>
<li>Chaux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+epidermal+growth+factor+receptor+is+frequently+overexpressed+in+penile+squamous+cell+carcinomas%3A+a+tissue+microarray+and+digital+image+analysis+study+of+112+cases%5BTitle%5D)+AND+%22Hum+Pathol%22%5BJournal%5D)">The epidermal growth factor receptor is frequently overexpressed in penile squamous cell carcinomas: a tissue microarray and digital image analysis study of 112 cases.</a> Hum Pathol. 44, 2690-2695 (2013).</li>
<li>McKenna, S. J., Amaral, T., Akbar, S., Jordan, L., Thompson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunohistochemical+analysis+of+breast+tissue+microarray+images+using+contextual+classifiers%5BTitle%5D)+AND+%22J+Pathol+Inform%22%5BJournal%5D)">Immunohistochemical analysis of breast tissue microarray images using contextual classifiers.</a> J Pathol Inform. 4, S13(2013).</li>
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</ol>

The authors have no disclosures

In this paper, a protocol for ngTMA is outlined. ngTMA is a newly established concept for tissue microarraying involving planning and design, digital pathology and slide annotation as well as automated tissue microarraying12.
Compared to conventional tissue microarraying, ngTMA offers many advantages. In a first step, the planning and design phase is critical. The focus is on answering a targeted research question. This should take into consideration the histological issues (e.g., which regions and how many spots do I want to include?), the statistical planning (e.g., sample size? How do I analyze my samples later?) and logistical considerations (e.g., how many biomarkers and therefore how many ngTMA copies?). A specific ngTMA is made for specific needs, whether it is for biomarker screening and high-throughput or intended to study specific histological aspects on only a few well&#160;selected cases.
One, if not the, most important disadvantage of conventional tissue microarraying is the low accuracy with which markings on the histological slides are made. The study of specific histological structures, cells or regions is made nearly impossible. ngTMA allows for high accuracy because annotations are placed directly on the digital slides. This allows the researcher to precisely select the regions to be punched, including specific cells. In this example, various areas within the same tissue block are punched out for arraying, such as the tumor center, the invasion front and areas of tumor/stroma interaction highlighted by the presence of small tumor cell clusters or even single cells. This accuracy can only be achieved using ngTMA. Thirdly, because slides are scanned onto a web-based digital platform, slide viewing and annotating can be made through the computer rather than with the microscope. The tissue arraying software provides a user&#160;friendly interface with a high degree of flexibility, therefore different layouts and ngTMA design can be achieved. Since TMA construction is performed automatically by drilling, there is little need for hands-on maneuvering and the amount of time for construction is significantly reduced. The time for TMA construction in this example here is between 24 hr. Using a conventional TMA approach and estimating 15 punches per hr, this project would take approximately 304 hr.
ngTMA can be applied to study protein expression, mRNA or DNA as well as combinations of these. Figure 9 illustrates several of these applications to potential biomarkers. Standard immunohistochemistry can be applied to determine the proliferation index of cancers using Ki-67. A combined approach to investigate protein expression and DNA amplification for genes such as HER2 can be used. Tissues can be gathered together on a single ngTMA to reduce costs, tissue usage and other resources. In addition, chromogenic mRNA in situ hybridization for HER2 and other genes can be performed to identify single mRNA transcripts in a large number of cases using a minimal number of tissue slides. Immunohistochemistry of immune markers such as CD8 can be visualized in the context of the tumor microenvironment. Double immunohistochemistry can also be used to highlight particular regions of interest such as interactions between immune cells (labelled in red) and tumor cells (labelled in brown) at the invasion front of cancers. Such a region of interest could not have been captured using conventional tissue microarraying.
Nonetheless, this protocol contains some limitations. The most important challenge is the overlap between donor block and digital slide. Several factors can influence this step. Firstly, the latest section from the block should be used for slide scanning. In many cases, the H&#38;E is not the last section of the donor block, rather it is an immunohistochemical or other stain. In this case, also a stained slide can be used as long as the last stain is scanned and annotated or a new H&#38;E should be made. Care should also be taken when tissue sections are being made as tissues may expand in the water bath which leads to challenging matching of slide and block. Secondly, at the moment projects are limited to 12 recipient TMA blocks processed at a single time. Larger projects exceeding 12 TMAs must be assigned to a second project name. Third, the donor blocks must be made using standard molds and cassettes as the instrument cannot adjust itself to various sizes. Finally, donor blocks must exceed the minimal height (4 mm) to achieve optimal drilling. In some cases, this requires reembedding of tissues.
Hundreds of publications over the last few years highlight the TMA as an invaluable tool for biomarker research. TMAs have been used to study lung13, colorectal7, breast14, prostate15, pancreas16, bladder17, and gastric18 cancers, to name a few. An increasing number of authors have combined the use of TMAs with image analysis and significant strides are being made in this direction19-21. However, beside a handful of research groups that have published innovative TMA ideas22-24, little attention has been given to optimize the TMA technique itself. Automated tissue microarrayers, such as the ATA-27 by Estigen/Beecher do provide layout design and expedient and automated tissue punching. This represents however only 1 aspect of the ngTMA concept.
ngTMA is a substantial improvement over conventional tissue microarraying techniques. It incorporates expertise in histology and TMA design with the flexibility of digital pathology and the precision of digital annotations with the speed and reliability of automated TMA construction. The combination of ngTMA and image analysis for evaluation of protein and molecular biomarkers will be a powerful tool to further improve the quality of clinical and translational research in the future.

Over the last two decades, tissue microarrays (TMAs) have had a remarkable impact on biomarker investigation studies. TMAs are essentially tissue &#8220;archives&#8221; produced by the repeated transfer of small tissue cores, typically ranging in size from 0.6 to 2.0 mm in diameter, from paraffin embedded &#8216;donor&#8217; blocks into a single TMA &#8216;recipient&#8217; block (Figure 1)1. Using a small core size, approximately 500 different tissue spots from few or many different patients can be arrayed into one TMA2.
The use of TMAs for prognostic or predictive biomarker studies has many advantages. Consider the example where expression of a protein biomarker by immunohistochemistry is to be evaluated on 450 patients. Rather than performing 450 immunohistochemical stains on 450 patient slides sectioned from the same number of blocks, small cores from each sample can be arrayed onto a single TMA block. Even if multiple cores are taken from each individual patient, a minimum number of blocks are produced. This has the considerable effect of drastically diminishing costs and other resources as well as reducing tissue wasting. Additionally this allows appropriately powered studies using a large number of tissues to be evaluated under the same experimental conditions.
TMAs have many different applications. For instance, they can be used to study morphology, protein expression, RNA expression and DNA aberrations following staining with different dyes, or after immunohistochemistry or chromogenic and even fluorescence in situ hybridization3-7. Recent studies have also used TMAs to test intra&#160;and interlaboratory variation in staining protocols, establish specificity or sensitivity of antibodies for specific gene mutations, and to determine the interobserver reproducibility of protein expression in international collaborations8-11.
The construction of traditional TMAs using patient-derived tissues is a long multi step procedure (Figure 2). It begins with a search for possible appropriate cases and selection of diagnostic slides from the archives at an institute of pathology, or other institute, from where they are retrieved. The pathologist evaluates each slide per case and selects the most representative slide for the purposes of the study. Next, the region of interest is marked using a pen directly under the microscope. This is often challenging and imprecise and results only in an &#8220;estimation&#8221; of where tissue punches should be taken from. Next, the paraffin blocks corresponding to these marked slides are retrieved from the archive. A quick comparison between block and slide is made. Using a semi-automated or homemade tissue arrayer, the donor block is punched out in the estimated region of interest and transferred into a recipient TMA block. Construction of TMAs using this arraying technique is labor intensive, time consuming, imprecise, and inflexible. Preparing a TMA of 475 spots in 3 copies is estimated to take about 84 hours of work.
A new approach to the construction of TMAs was recently introduced by the Institute of Pathology, University of Bern that relies on three components: planning and design (or consulting), digital pathology combined with expertise in histology and automated TMA arraying12. Together, this concept is called next generation Tissue Microarray (ngTMA). Below, a protocol for ngTMA is described based on an example of 134 patients with metastatic colorectal cancer. Here, primary tumors as well as lymph node metastases and liver metastases are to be arrayed into ngTMAs for subsequent biomarker analysis. Additionally, small tissue cores from each patient are desired for future nucleic acid extraction.

<table><tbody><tr><td>Pannoramic 250 Flash II</td><td>3DHistech</td><td></td><td>Slide scanner</td></tr><tr><td>TMA Grandmaster</td><td>3DHistech</td><td></td><td>Tissue Microarrayer</td></tr><tr><td>Pannoramic Viewer</td><td>3DHistech</td><td>free</td><td>Slide viewing software</td></tr><tr><td>Case Center</td><td>3DHistech</td><td></td><td>Digital slide platform</td></tr></tbody></table>

a next-generation tissue microarray (ngtma) protocol for biomarker studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

Planning and Design
Here the example of 134 patients with metastatic colorectal cancer that are being investigated for a particular series of biomarkers was used. Not only is an ngTMA planned for these patients, but DNA extraction followed by mutational analysis for some specified gene.
During the planning and design phase of ngTMA, the following aspects have been decided. From each patient, 6 different histological regions will be investigated: tumor center, tumor invasion front, areas of densest tumor budding (tumor/stroma interaction), normal tissue, lymph node metastases and liver metastases. Three tumor spots per tumor tissue area and two normal tissue spots are to be included into the project (n=17 spots per patient). Since it is planned that more than 50 biomarkers be investigated on this patient material, two copies of the final ngTMA are desired. Due to the possible small size of lymph node and distant metastases, a small core diameter of 0.6 mm for TMA construction for all tissues is chosen.
Additionally, it is planned that each region be separately arrayed, so that 6 ngTMAs containing different histological areas will be produced: an ngTMA containing tissues from the a) tumor center, b) invasion front, c) tumor budding areas, d) normal tissue, e) lymph node metastases, and f) liver metastases.
For the TMA design using a 0.6 mm tool, a comfortable distance of 0.4 mm between punches will be selected. Because evaluation of tissue spots in long rows and columns is often tiresome, two different designs are selected. For tumor blocks with 3 punches per tissue area, a design containing six parts is created. This leads to a total number of 402 tissue punches. For normal tissue punches, 2 punches per case are to be included. A layout fitting 432 punches in total is chosen.
Moreover, for each case, up to 4 cores at 0.6 mm will additionally be punched out and placed into 0.2 ml tubes. These tissue cores will also be annotated.
To summarize, 6 ngTMAs of different tissues will be produced (5 tumor arrays x 3 punches x 134 patients; 1 normal tissue array x 2 punches x 134 patients) in addition to 4 cores per patient for tubes. Taken together, 2814 annotations will be made.
Next, the corresponding H&#38;E slides from selected patients are retrieved from the slide archive. Each case is briefly reviewed by a pathologist and most representative slides of each tissue type are selected.
Scanning
Digital slides of each histological section are made for future annotation. For this study, 3-4 tissue slides are to be scanned per case, i.e., primary colorectal cancer, with or without adjacent normal tissues, lymph node metastases and liver metastases and possibly an additional slide containing normal mucosa.
Up to 10 different magazines can be fully loaded into the slide scanner; therefore 250 slides can be scanned in a single run. Time for scanning and digital slide size varies with the dimensions of the tissue and the quality factor desired. The quality factor ranges from 0 to 100. Here, a quality factor of 60 is selected, since it produces a smaller scan file size without any noticeable loss of scan quality. Small biopsies can be scanned in under 30 sec while whole tissue sections, like the ones used in this example may take between 2 and 10 min. Digital slide size can also range from 2 MB to 2 GB. Using a quality factor of 60, scans between 600 MB and 1 GB are produced.
Between 402 and 536 slides are required for this project totaling 3 scanning runs. Each run is performed overnight. Approximately 500 GB of storage space is needed. Slides are scanned directly onto a digital platform called Case Center (ngtma.unibe.pathcasecenter). Case Center is accessible from anywhere with web access by entering the designated username and password.
Annotation
For annotation, two aspects should be considered: 1) different tissue areas should be given different annotation colors and 2) the number of annotations should reflect the number of desired copies of the final ngTMA.
In this case, 3 spots per tumor area for a single copy have been selected. For 2 copies, each region must be annotated using 6 spots. Useful are green for normal tissue, blue for the tumor center, yellow for the tumor invasion front, red for areas of tumor budding (tumor/stroma) interaction, black for lymph node metastases, turquoise for distant metastases and finally white for regions to be used for tubes. Annotation of each case requires 2-3 min.
ngTMA Construction
This study requires 6 ngTMA blocks to be constructed in 2 copies, resulting in 12 final blocks. The maximum number of recipient blocks that can be supported by the arrayer is 12. Each tumor block will contain 402 tissue spots and each normal tissue block will contain 268 spots, totaling 4,556 spots across the entire project.
A TMA layout is created for each recipient block and the project is saved (Figures 6A, 6B). 60 tissue blocks can be loaded into the tissue microarrayer in parallel; an image of each donor block is taken. Starting with the first donor block, the corresponding annotated digital slide is retrieved from Case Center. Slide matching is performed, which can take between 1-3 min. Annotations are confirmed and the arrayer is prompted to start punching (Figure 7). Core punching and transfer time is approximately 12 sec. Loss of cores during this procedure is minimal. Total time for arraying this project is approximately 24 hr, including time for slide/block matching and reloading of the instrument. Some examples of ngTMA are illustrated in Figure 8A. Cores for subsequent molecular analysis can also be punched out at this time (Figure 8B).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig1highres.jpg" width="500" /> 
Figure 1.&#160;A) A &#8216;donor&#8217; block. Cores from the donor block are punched out and transferred into B) a recipient block, or tissue microarray (TMA).
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig2highres.jpg" width="500" /> 
Figure 2.&#160;The conventional tissue microarray workflow. A) Histological slides are retrieved from the archive and B) given to the pathologist for review. C-D) Annotations are made on the slides indicating the &#8216;estimated&#8217; region for transfer. E) The corresponding tissue blocks are retrieved. F) Block and slide are compared for matching, which can G) sometimes be a challenging task. H-J) Tissue microarraying then takes place. K) Cores are repeatedly transferred and L) a final TMA is constructed.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig3highres.jpg" width="500" /> 
Figure 3.&#160;A) Up to 25 slides can be placed in each magazine for scanning. B) Adjustments to the quality factor and size can be made. Scanning progress can be monitored.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51893/51893fig4highres.jpg" width="600" /> 
Figure 4.&#160;A) Scanned slides can be uploaded onto the web-based platform, Case Center. B) Digital slides can be viewed. The TMA annotation tool allows the user to select the core size and color required for annotation. C) Annotations can be placed directly onto the digital slide. <a href="https://www.jove.com/files/ftp_upload/51893/51893fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig5highres.jpg" width="500" /> 
Figure 5. A) Up to 60 blocks can loaded into the tissue microarrayer, namely 10 donor blocks per row and 12 recipients blocks as well. B) An image of each donor block is taken, the digital slide is retrieved. Matching of block and slide is done and annotations are used for block punching. C) The progress of the arraying can be monitored while the instrument is punching. D) After punching is performed, an export of the location of each spot in the layout but also an image of each donor block with corresponding annotations is made.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig6highres.jpg" width="500" /> 
Figure 6. These TMA layouts were used for arraying the A) tumor blocks and B) normal tissues.
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig7highres.jpg" width="500" /> 
Figure 7. Screenshot of the tissue microarrayer software. On the left, 12 recipient blocks can be placed in the instrument. On the bottom, images of each donor block are taken. In the center, the TMA layout, the progress of the punching as well as enlarged images of each donor block with annotations is found.
<img alt="Figure 8" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig8highres.jpg" width="500" /> 
Figure 8.&#160;A) Some examples of newly created ngTMA blocks. B) Additional tissue punches can be taken for subsequent molecular analysis. These cores are punched out and placed in 0.2 ml tubes.
<img alt="Figure 9" fo:content-width="6in" src="/files/ftp_upload/51893/51893fig9highres.jpg" width="600" /> 
Figure 9. Examples of ngTMA biomarker applications. A) Immunohistochemistry staining of Ki67 in colorectal cancer. Dual HER2 immunohistochemistry/in situ hybridization assay showing B) low expression of protein (brown) and no amplification of the Her2 gene (black) relative to the centromere (red); C) high expression of the protein (brown) and Her2 gene amplification (black) relative to centromere (red). D) Chromogenic in situ hybridization of Her2 showing single mRNA transcripts in breast cancer ngTMA. E) Immunohistochemistry for CD8 in colorectal cancer. F) Double immunohistochemistry stain for pan-cytokeratin marker AE1/AE3 (brown; tumor cells) and CD68 (red; macrophages) highlighting tumor microenvironment in colorectal cancer. <a href="https://www.jove.com/files/ftp_upload/51893/51893fig9highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies at JoVE.com

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

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Research

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Medicine

24.0K Views.  University of Bern. The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

Video: A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer

The tissue microarray (TMA) is an important research tool in which many formalin fixed paraffin embedded (FFPE) samples can be represented in a single paraffin block. This is achieved by using tissue cores extracted from the region of interest of different donor FFPE blocks and arranging them into a single TMA paraffin block. Once constructed, sections from the completed TMA can be used to perform immunohistochemistry, chromogenic, fluorescence in situ hybridization (FISH) and RNA ISH studies to assess protein expression as well as genomic and transcriptional alterations in many samples simultaneously, thus minimizing tissue usage and reducing reagent costs. There are several different TMA construction techniques. One of the most common construction methods is the recipient method, which works best with cores of the same length for which a minimum length of 4 mm is recommended. Unfortunately, tissue blocks can be heavily resected during the diagnostic process, frequently resulting in "non-ideal" donor block thicknesses of less than 4 mm. The current article and video focus on the double-sided adhesive tape method; an alternative manual, low cost, easy to use, and rapid method to construct low density (&lt;50 cores) TMAs that is highly compatible with these non-ideal donor blocks. This protocol provides a step-by-step guide on how to construct a TMA using this method, with a focus on the critical importance of pathological review and post construction validation.

This protocol outlines the tape method on how to manually construct a tissue microarray using FFPE donor blocks of differing depths.

The tissue microarray (TMA) is an important research tool in which many formalin fixed paraffin embedded (FFPE) samples can be represented in a single ...

Cancer Research

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Bioengineering

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.

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 ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

Immunology and Infection

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Biology

Predictive Immune Modeling of Solid Tumors

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment response challenging. The ability to resolve the relative populations of immune cells present in and around the tumor tissue has been shown to be clinically-relevant to understanding response, but is limited by traditional techniques such as flow cytometry and immunohistochemistry (IHC), due the large amount of tissue required, lack of accurate cell type markers, and many technical and logistical hurdles. One assay (e.g., the ImmunoPrism Immune Profiling Assay) overcomes these challenges by accommodating both small amounts of RNA and highly degraded RNA, common features of RNA extracted from clinically archived solid tumor tissue. The assay is accessed via a reagent kit and cloud-based informatics that provides an end-to-end quantitative, high-throughput immuno-profiling solution for Illumina sequencing platforms. Researchers start with as few as two sections of formalin-fixed paraffin-embedded (FFPE) tissue or 20-40 ng of total RNA (depending on sample quality), and the protocol generates an immune profile report quantifying eight immune cell types and ten immune escape genes, capturing a complete view of the tumor microenvironment. No additional bioinformatic analysis is required to make use of the resulting data. With the appropriate sample cohorts, the protocol may also be used to identify statistically significant biomarkers within a patient population of interest.

The use of an RNA-based approach to determine quantitative immune profiles of solid tumor tissues and leverage clinical cohorts for immune-oncology biomarker discovery is described through a molecular and informatics protocols.

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment ...

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue microarrays make up the majority of multi-tissue paraffin blocks, but increasingly, researchers are using non-arrayed blocks containing larger tissues from multiple individuals which can provide many of the advantages of tissue microarrays without substantial investment in planning and equipment. A critical component of any multi-tissue analysis is the orientation method used to identify each individual tissue. Although methods exist to maintain proper orientation and identification of tissues in multi-tissue blocks, most are not well-suited to non-arrayed blocks, may consume valuable space within an array and/or are difficult to produce in the standard histology laboratory. The Specimen Orientation Tag (SpOT) is a simple, low cost orientation tool that is clearly visible in paraffin blocks and all tissue sections for reliable specimen identification in arrayed and non-arrayed layouts. The SpOT provides advantages over existing orientation methods for non-arrayed blocks as it does not require any direct modification to the tissue and allows for flexibility in the arrangement of tissue pieces.

The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections.

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...