TissueScan qPCR Array FAQs

TissueScan Tissue qPCR Arrays are panels of pre-normalized cDNA from multiple (48 for most of the panels) cancer tissues. The tissue samples used for each type of Tissue qPCR Array cover all disease stages and are accompanied by comprehensive pathology reports. By designing a pair of gene-specific or SNP specific primers, scientists can study the following via a single real-time PCR reaction

• Expression profile changes during disease progression for a particular transcript
• SNP profiling throughout disease progression
• Validation of biomarker candidates obtained from microarray data
• Relevance of a potential tumor suppressor/oncogene across different cancer types

OriGene has a growing list of covered diseases that are shown in the website.

All of the frozen tissues were flash frozen within 60 minutes of ischemia. The majority of our samples were frozen within 30 minutes and many within 15 to 20 minutes. All of the RNA is examined before shipping and shows minimal to no degradation by Agilent Bioanalyzer analysis. An electropherogram indicating RNA quality is provided for each sample.

No, the tissue was not micro-dissected to separate tumor from non-tumor cells. Samples were selected based on tumor content
(minimally 50% tumor) as determined by microscopic pathology analysis. Such information is available for each sample in the pathology report.

The tissue samples are frozen in OCT, which serves as an excellent protectant against the effects of long term '80C storage. Before processing the tissue for RNA isolation, most of the frozen OCT is dissected away prior to homogenization. Also, reagents and protocols for RNA isolation have been optimized to overcome residual OCT interference.

The G?￡normalG?￥ tissues are taken from patients diagnosed with a tumor, but the tissues harvested were from normal regions.
Another term for these tissues would be disease stage

Some of the normal, or disease stage 0, samples were taken from patients from whom other tissue samples (disease stages I-IV) were taken. These paired samples can be identified by the identical pathology report numbers listed for different wells.

We received high quality total RNA from Cytomyx and generated 1^st strand cDNA at OriGene using an oligo-dT primer. Cytomyx provides an in-depth pathology report (including histology sections) for all of the RNA used in these panels, which can be viewed on OriGene's website.

Well to well amounts of cDNA may vary due to normalization based on beta-actin. The average amount of cDNA is 2-3 ng per well, corresponding to 2-3ng of mRNA.

• For the colon cancer array, we tested the expression of human carbonic anhydrase II (CA2) (NCBI accession # NM_000067) and found expression levels to be downregulated in non-normal tissues.
• For the lung cancer array, we tested the expression of human topoisomerase (DNA) II alpha 170kDa (TOP2A) (NCBI accession # NM_001067) and found expression levels to be upregulated in non-normal tissues.
• For the ovarian cancer array, we tested the expression of human kallikrein 8 (neuropsin/ovasin) (KLK8), transcript variant 1 (NCBI accession # NM_007196) and found expression levels to be upregulated in non-normal tissues.
• For the thyroid cancer array, we tested the expression of human dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2) (DPP4) (NCBI accession # NM_001935) and found expression levels to be upregulated in non-normal tissues.
• For the breast cancer array, we tested the expression of human HER2 (NCBI accession # NM_001005862) and found expression levels to be upregulated in HER2 responsive patient tissues.
• For the lymphoma, liver, melanoma, endometrium, gastroesophageal, cancer survey, colon cancer II and other arrays, we tested the expression of human Survivin (NCBI accession # NM_001168) and found expression levels to be upregulated in non-normal tissues.
• For the prostate cancer array, we tested the expression of human hepsin (Hep) (NCBI accession # NM_182983) and found expression levels to be upregulated in non-normal tissues.
• For the kidney cancer array, we tested the expression of human hepsin (Hep) (NCBI accession # NM_182983) and found expression levels to be upregulated in non-normal tissues.
• For the Crohns and Colitis array, we tested the expression of human tumor necrosis factor (TNF, NCBI accession # NM_000594) and found expression levels to be upregulated in non-normal tissues.

The plate is in a 12x8 array in a 96 well plate. The 48 samples begin on row C, column 1 and continue to row F, column 12. However, please see the Certificate of Analysis that accompanies your sample for a layout diagram or download the layout from the website.

We frequently use Applied Biosystems (http://www.appliedbiosystems.com ) as our source of real-time PCR reagents (buffers, etc.), and order primers from MWG Biotech (http://www.mwg-biotech.com/html/all/index.php) or BBL (http://www.bioserve.com/index.cfm ), both of whom offered HEX-labeled primers.

The actin sequence used to design the primers was NCBI accession # NM_001101, for Homo sapiens actin, beta (ACTB), mRNA.
The primer sequences and targeted actin sequences are as follows:

• forward primer: CAGCCATGTACGTTGCTATCCAGG
• reverse primer: AGGTCCAGACGCAGGATGGCATG
• targeted beta-actin sequence: CAGCCATGTACGTTGCTATCCAGGCTGTGCTATCCCTGTACGC CTCTGGCCGTACCACTGGCATCGTGATGGACTCCGGTGACGGGGTCACCCA
  CACTGTGCCCATCTACGAGGGGTATGCCCTCCCCCATGCCATCCTGCGTCTGGACCT

Each Taqman probe may have its optimal working concentration. You can determine it by doing some pilot experiments. However according to Applied Biosystems protocol, a 1x or 250 nM final concentration usually gives good results.

TissueScan are made with PCR plates from Abgene (AB-0600). According to the plate manufacturer, the plates will fit in the following machines:
Standard Thermocyclers

• Biometro Uno, Uno II, T1, Tgradient, TRobot
• BioRad iCycler and MyCycler
• Eppendorf Mastercycler Gradient and Mastercycler EP Gradient
• Ericomp SingleBlock system, TwinBlock system, and Deltacycler I
• ThermoHybaid PCR Express, Px2, PxE, MultiBlock System and MBS, Touchdown, Omnigene, and Omn-E
• MJ Research PTC-200 DNA Engine, PTC-225 DNA Tetrad/PTC-220/221 DNA Dyad, and PTC-100 with 96 well block
• MWG Primus 96 and TheQ Lifecycler
• ABI GeneAmp 2700/2720, GeneAmp 9600, and GeneAmp 9700
• Stratagene Robocycler
• TaKaRa TP3000
• Techne TC-412/512, Touchdown Gradient, Flexigene, and Genius

qPCR thermocyclers

• ABI Prism 7000, 7700, 7300, 7500, and 9500 (except fast block formats)
• BioRad iCycler and MyiQ
• Stratagene MX4000, MX3000p, and MX3005p

If your thermocycler is not listed, please contact Technical Support for more information. Another solution (not recommended) is to resuspend the cDNA in the provided plate and transfer it to a plate compatible with your machine. To do so, add half of the reaction volume to each well of the product plate and incubate on ice for 20 minutes. Then transfer the resuspended solution to a plate using a multi-channel pipette, add the remainder of the reaction volume and other reagents, and run your experiment. Exceptionally careful resuspension and pipetting would be mandatory to preserve the quantitative nature of this product, which is why this option is not recommended.

OriGene is working on monthly releases of new Tissue qPCR Arrays. Please visit our website regularly for the most up to date information. OriGene also appreciates your ideas and suggestions for development of future panels.

Please use the product name (e.g. TissueScan Breast Tissue qPCR Array) followed by OriGene Technologies, Rockville MD. If you wish, you can drop us an email and we would be happy to list your paper in our citations page.

Our repository contains an extensive collection of both pathology confirmed normal and diseased tissue samples. For many diseases, we are even able to provide patient matched diseased and normal tissue.

Our partner, CYTOMYX believes that the proper collection and use of human biospecimens begins with the complete protection of the rights and privacy of the individual, thus all samples are collected under IRB approved protocols. Moreover, all human subjects are fully informed and are explicitly asked for their consent to future research use of their samples, even in cases where such uses are unknown at the time. All samples are de-identified and coded with patient numbers to ensure the privacy of our donors in accordance with the Health Insurance Portability and Accountability Act (HIPAA).

Given the potentially heterogeneous nature of a given tissue sample, we cannot guarantee that the sample cellularity will remain constant throughout the entire tissue block. Instead, our histologists prepare an H&E section from each individual sample that is then examined by our pathologists in order to determine the specific cellularity of that sample (e.g., percent tumor cells, percent normal cells, etc.) in order to assign that tissue an accurate sample level pathology diagnosis. The presence of infiltrating macrophage, for example, is often noted in the standard data set that accompanies each tissue sample.

Each sample is accompanied by a rich data set presented in tabular format. The spreadsheet includes (but is not limited to): patient age, patient gender, tissue of origin/finding, patient level diagnosis, sample level diagnosis, tumor grade, TNM classification, minimum stage grouping, detailed sample cellularity (% tumor cells, % normal cells, % stroma, % necrosis) and pathology verification notes (when available). The spreadsheet also includes hyperlinks to detailed abstractions of the patients' pathology reports (which often contain diagnostic findings beyond what is presented in the spreadsheet) and hyperlinks to digital H&E images (4x and 20x magnification) for each sample.

Since each individual tissue sample is examined by a pathologist, we are able to assign values for the percent of normal, lesion, tumor, tumor hypercellular stroma, tumor hypo/acellular stroma and necrosis based on Pathology Verification, a process of sample validation that relies on microscopic examination of the H&E slide for a given sample. A more detailed description of each term is provided below:

• Normal - Percent distribution of normal cells in the sample.
• Lesion - Percent distribution of lesional cells. Lesional cells are neither cancerous, nor are they described as "within normal limits". An example of a lesion lung cell is one that is best described as exhibiting emphysematous changes (i.e., not cancer, but not entirely normal either).
• Tumor - Percent distribution of tumor cells in the sample.
• Tumor Hypercellular Stroma - Percent distribution of tumor hypercellular stroma cells.
• Tumor Hypo/Acellular Stroma - Percent distribution of tumor hypo-/acellular stroma cells in the sample.
• Necrosis - Percent distribution of necrotic cells in the sample.

For some of our patient cases, we are able to provide additional clinical information beyond what is presented in the tabular spreadsheet and abstracted pathology report. Such data may include treatment history, pre-op medications, pre-op lab test results and outcomes and is only available upon request. If you are interested in obtaining additional clinical data for any of our samples, please contact our technical support team

In many cases, both diseased and normal adjacent tissues have been banked from the same surgical event. The likelihood that a normal adjacent tissue sample is collected in conjunction with a diseased tissue sample from the same patient, however, can depend largely on the surgical procedure being performed. Though normal tissue is not strictly targeted for banking (except in rare circumstances), it is often obtained as part of the surgical event. Thus, for some patient cases, both diseased and pathology confirmed normal tissues are available, but for others, only diseased OR only normal tissue has been banked.

Here is our guideline:

Full Product Name, OriGene Technologies Inc., Rockville, MD, USA, Catalog #, Lot #.

In addition, we would love to hear from you when the paper is published. Email us a copy of the accepted manuscript and receive a special gift.