Name: Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR (Video) | JoVE Journal
Uploaded: 2016-01-29T00:00:00.000+00:00
Duration: 8 min 56 s
Description: Learn quantitative real-time PCR to detect and quantify tumor-specific mRNA in mouse lung tissue for analyzing metastasis. This method provides sensitive measurement of cancer progression in mammary tumor models.

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5 minutes

The Yeh lab is supported by research funding from an American Cancer Society Institutional Research Grant (IRG-97-219-14) awarded to the Hollings Cancer Center at MUSC, by research funding from a Department of Defense grant (W81XWH-11-2-0229) at MUSC, and by an award from the Concern Foundation (to ESY).

The protocol follows the guidelines and animal care standards of the Medical University of South Carolina (MUSC) and its Institutional Animal Care and Use Committee (IACUC).
1. Lung Dissection
<ol>
	<li>Mammary tumor dissection (optional depending on study goals and whether primary tumor analysis or tail vein injection was performed).
	<ol>
		<li>Euthanize mouse using a lethal dose of isoflurane anesthesia according to IACUC and university regulation. Confirm euthanization by surgical dislocation.&#160;</li>
		<li>On foam board, pin with dissection pins by placing mouse on its back with limbs spread.</li>
		<li>Spray mouse with 70% ethanol.</li>
		<li>Using forceps, grasp the lower portion of the animal at the center.</li>
		<li>Using scissor, cut upward toward the neck through the skin, being careful not to pierce the peritoneal cavity of the mouse.</li>
		<li>Cut back skin at the limbs to reveal tumor tissue.</li>
		<li>Dissect out tumor tissue and preserve by preferred method, such as freezing or fixation, for further analysis (not discussed further in this protocol).&#160;</li>
	</ol>
	</li>
	<li>Lung tissue dissection.
	<ol>
		<li>If tumor tissue was harvested as described above, pin down any excess skin.</li>
		<li>Using forceps, grasp peritoneum at lower end of the animal and begin cutting upwards toward the base of the neck.</li>
		<li>Carefully cut along the left and right sides of the rib cage being careful to avoid severing any blood vessels that may bleed into the thoracic cavity. Remove the rib bones by cutting to the left and right through the diaphragm and upper portions of the rib cage.</li>
		<li>Using forceps grasp the trachea of the mouse and pull forward.</li>
		<li>Sever the trachea with dissection scissors and remove lungs from mouse. 
		Note: The heart may remain attached to the lung. If this occurs, carefully dissect heart away from lungs being careful to collect all five lobes (see Figure 1).</li>
		<li>Wash gently with PBS to remove excess blood.</li>
	</ol>
	</li>
	<li>Gross evaluation of lung tissue.
	<ol>
		<li>Option 1: In vivo imaging.
		<ol>
			<li>For luciferase imaging, ~10 min prior to imaging, give animals a 150 mg/kg dose of luciferin by intraperitoneal (i.p.) injection. Image lungs in vivo in anesthetized animals or upon removal after dissection (Figure 2) 5.</li>
		</ol>
		</li>
		<li>Option 2: Gross evaluation.
		<ol>
			<li>Examine lungs under stereo microscope to visualize tumor nodules. Note: If cells are GFP labeled, a fluorescence capable stereomicroscope containing a light cube with the appropriate excitation/emission maxima (e.g. near 470/510 nm) to detect GFP may be used to examine lungs stereoscopically for fluorescence prior to processing.</li>
		</ol>
		</li>
		<li>If lungs are to be analyzed immediately, proceed to &#39;RNA Isolation&#39; section. Otherwise, snap freeze tissue using liquid nitrogen or dry ice. Store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. RNA Isolation
Note: For the representative analysis, RNA was isolated using a RNA isolation kit (see Materials List). While the current analysis uses one specific manufacturer&#39;s product, a variety of isolation kits are available from several reputable vendors. Additionally, non-kit based centrifugation methods are also an option. cDNA should also be prepared from a positive control RNA sample, such as a cell line or plasmid, for standard curve analysis. Alternatively, a positive control specific for the primer probe set may be purchased (see Materials List).
<ol>
	<li>If using previously frozen tissue, gather frozen tissue on dry ice.</li>
	<li>Homogenize tissue using one of the following methods such as: mortar and pestle grinding performed by hand over dry ice after freezing tissue in liquid nitrogen; tissue homogenizer or mechanical dissociation device per manufacturer&#39;s suggestion; sonication, or other preferred method. 
	Note: For the representative analysis, the preferred method of homogenization is sonication.
	<ol>
		<li>For the analysis presented in Figure 3, prepare lysis buffer (provided in RNA isolation kit) and 2-mercaptoethanol at a ratio of 20 &#181;l 2-mercaptoethanol for every 1 ml of lysis buffer. Resuspend tissue in 300 &#181;l lysis buffer supplemented with 2-mercaptoethanol for every 30 g of tissue and sonicate for 10 sec with sonicator set at 30% amplitude.</li>
		<li>If using mortar and pestle, resuspend ground tissue in 300 &#181;l lysis buffer after grinding.</li>
	</ol>
	</li>
	<li>Add 10 &#181;l proteinase K to 590 &#181;l TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). Add 600 &#181;l proteinase K solution to every 300 &#181;l (i.e. for every 30 mg) of tissue. Incubate for 10 min at RT. 
	Note: Digestion time may vary.&#160;&#160;</li>
	<li>Centrifuge samples at &#8805;13,000 g for 10 min to remove debris.</li>
	<li>Transfer supernatant to new tubes.</li>
	<li>Add 450 &#181;l ethanol (96%-100%) for every 900 &#181;l of supernatant to precipitate RNA.</li>
	<li>Transfer 700 &#181;l of the lysate/ethanol mix to the column.</li>
	<li>Centrifuge samples at &#8805;13,000 g for 1 min to bind RNA to column.</li>
	<li>Discard liquid waste and add remaining supernatant to column and spin again.</li>
	<li>Once all the lysate is spun through the column, add 350 &#181;l of Wash Buffer 1 to the upper portion of the column and centrifuge samples at &#8805;13,000 g for 30 sec.</li>
	<li>Discard liquid from column and replace upper portion.</li>
	<li>Prepare DNase by mixing 5 units of DNase I enzyme with 5 &#181;l of 10x DNase and 40 &#181;l of DNase/RNase free water per sample (total volume of 50 &#181;l/sample).</li>
	<li>Add 50 &#181;l of DNase mix to each column and incubate for 15 min at RT.</li>
	<li>Add 350 &#181;l of Wash Buffer 1 to column and centrifuge samples at &#8805;13,000 g for 30 sec.</li>
	<li>Discard liquid from column and replace upper portion.</li>
	<li>Add 600 &#181;l of Wash Buffer 2 to the column and spin 30 sec at &#8805;13,000 g.</li>
	<li>Discard liquid from column and replace upper portion.</li>
	<li>Add 250 &#181;l of Wash Buffer 2 and centrifuge for 2 min at &#8805;13,000 g.</li>
	<li>Put part of column into new collection tube and discard old collection tube.</li>
	<li>Add 50-100 &#181;l of RNase/DNase free water to column and incubate for 1 min.</li>
	<li>Spin for 1 min at &#8805;13,000 g.</li>
	<li>Re-run collected eluate through column to increase RNA yield.</li>
	<li>For immediate use, keep samples on ice and proceed to quantitation. For use later, snap freeze on dry ice or liquid nitrogen. Store at -80 &#176;C freezer until needed.</li>
</ol>
3. First Strand Synthesis
Note: For the representative analysis, the reverse transcriptase (RT) reaction was performed a First Strand cDNA synthesis kit designed for QRT-PCR (see Materials List).
<ol>
	<li>If samples were previously collected, thaw tubes on ice.</li>
	<li>Measure RNA quantity using a spectrophotometer.</li>
	<li>Using 0.5-2 &#181;g of total RNA, perform first strand synthesis using reverse transcriptase to generate cDNA stock.
	<ol>
		<li>In sterile, RNase/DNase-free PCR tubes/plates, prepare the reaction mix (Table 1). Mix gently and centrifuge. Program a standard PCR machine (Table 2).</li>
	</ol>
	</li>
	<li>Dilute finished reaction to desired volume (generally 50-100 &#181;l) using sterile nuclease-free water.</li>
</ol>
4. Real-time PCR
Note: For the representative analysis, SYBR green was used. However, any preferred method can be substituted at the user&#39;s discretion.
<ol>
	<li>Using a positive control cDNA, set up a standard curve for each primer set.
	<ol>
		<li>For the analysis shown in Figure 3, prepare the standard curve for human HER2 using the manufacturer recommended positive control cDNA (see Materials List). For mouse gapdh, use the negative control lung cDNA to generate a standard curve. For each probe, serially dilute the cDNA 1:4 to generate 5 standards.</li>
	</ol>
	</li>
	<li>Calculate the number of total samples, which should include the standard curve and experimental samples. 
	Note: For the analysis described here, 2-3 experimental replicates per sample were analyzed. Standard curve analysis was performed to generate a simple linear regression model that can be applied to determine the relative amount of HER2 and gapdh per sample to calculate final normalized quantity. This analysis will also ensure that the primer efficiency is adequate for the analysis at hand.</li>
	<li>In a sterile, RNase/DNase-free tube, prepare master mix (Table 3).
	<ol>
		<li>Calculate master mix amount per sample. Scale up for more than one sample. Use a master mix for each experimental gene of interest, as well as an internal control such as gapdh. Use primers for gapdh at a final concentration of 200 nM. 
		Note: The internal control is particularly useful when trying to distinguish between human and mouse cell origin. 
		Note: Primer concentration for HER2 was optimized and determined by the manufacturer.</li>
	</ol>
	</li>
	<li>Prepare test plate containing 1 &#181;l cDNAs corresponding to each standard or experimental sample. Allocate at least 1 cDNA sample/well for each primer probe. Add 19 &#181;l of master mix per well for each primer probe. 
	Note: For the representative data in Figure 3, the cDNA samples were analyzed in duplicate for each primer probe and graphed as the average of the two samples.</li>
	<li>Run reaction in QRT-PCR machine using recommended or previously tested PCR protocol. See Table 4 for the PCR protocol used for the representative data in Figure 3 and Figure 4.&#160;</li>
</ol>

<ol><li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+metastasis%3A+building+a+framework%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Cancer metastasis: building a framework.</a> Cell. 127 (4), 679-695 (2006).</li>
<li>Bos, P. D., Nguyen, D. X., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+metastasis+in+the+mouse%5BTitle%5D)+AND+%22Curr+Opin+Pharmacol%22%5BJournal%5D)">Modeling metastasis in the mouse.</a> Curr Opin Pharmacol. 10 (5), 571-577 (2010).</li>
<li>Kim, I. S., Baek, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+for+breast+cancer+metastasis%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Mouse models for breast cancer metastasis.</a> Biochem Biophys Res Commun. 394 (3), 443-447 (2010).</li>
<li>Saxena, M., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rebuilding+cancer+metastasis+in+the+mouse%5BTitle%5D)+AND+%22Mol+Oncol%22%5BJournal%5D)">Rebuilding cancer metastasis in the mouse.</a> Mol Oncol. 7 (2), 283-296 (2013).</li>
<li>Yang, S., Zhang, J. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+for+tumor+metastasis%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Mouse models for tumor metastasis.</a> Methods Mol Biol. 928, 221-228 (2012).</li>
<li>Rampetsreiter, P., Casanova, E., Eferl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+modified+mouse+models+of+cancer+invasion+and+metastasis%5BTitle%5D)+AND+%22Drug+Discov+Today+Dis+Models%22%5BJournal%5D)">Genetically modified mouse models of cancer invasion and metastasis.</a> Drug Discov Today Dis Models. 8 (2-3), 67-74 (2011).</li>
<li>Condeelis, J., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+imaging+of+cell+movement+in+tumours%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Intravital imaging of cell movement in tumours.</a> Nat Rev Cancer. 3 (12), 921-930 (2003).</li>
<li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+imaging+in+living+subjects%3A+seeing+fundamental+biological+processes+in+a+new+light%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17 (5), 545-580 (2003).</li>
<li>Zinn, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+bioluminescence+imaging+in+small+animals%5BTitle%5D)+AND+%22ILAR+J%22%5BJournal%5D)">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49 (1), 103-115 (2008).</li>
<li>Tseng, J. C., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+bioluminescence+imaging+of+mouse+tumor+models%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Quantitative bioluminescence imaging of mouse tumor models.</a> Cold Spring Harb Protoc. 2015 (1), (2015).</li>
<li>Radonic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guideline+to+reference+gene+selection+for+quantitative+real-time+PCR%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Guideline to reference gene selection for quantitative real-time PCR.</a> Biochem Biophys Res Commun. 313 (4), 856-862 (2004).</li>
</ol>

ESY and MAA are not employed by FirstString Research Inc. and do not hold any financial interest in the company. FirstString Research Inc. provided the luciferase imaging data that was presented in this manuscript. GSG is president and CEO of FirstString Research. CLG is an employee of FirstString Research. GSG and CLG have stock options issued by FirstString Research.

This protocol describes use of QRT-PCR to evaluate mammary tumor cell metastasis to the lung using a xenograft mouse model. It is acknowledged that other techniques, including bioluminescent imaging, are available and are also effective for detecting metastasis despite having their own values and limitations. The data presented suggests that QRT-PCR provides an effective measure of detecting tumor-derived mRNA within lung tissue for quantitation of total metastasis. It is proposed that QRT-PCR analysis provides a seconda...

QRT-PCR analysis is proposed as a method for assessing tumor metastasis. This method is proposed as an alternative for users that are interested in evaluating metastasis but may not have access to specific equipment such as in vivo imaging equipment or a fluorescence capable stereoscope. A discussion of commonly used methods is presented followed by a demonstration for how QRT-PCR analysis can be used either as a separate or as a companion method to evaluate metastasis. This procedure has the potential to provide a quantitative analysis of metastatic burden.
Standard methods of gross analysis, including visualization of lungs under a stereomicroscope as well as serial sectioning followed by hematoxylin and eosin (H&#38;E) staining of lung tissue, are quantifiable but rely heavily on user defined parameters for counting 2-5. When evaluating whole lungs using a stereomicroscope, only large surface metastases are visible and analysis requires the investigator to have reasonable knowledge of lung anatomical structure to determine what constitutes a metastatic lesion. Fluorescent labeling of tumor cells with a marker such as GFP and use of a stereomicroscope that contains a light cube with the appropriate excitation/emission maxima (e.g. near 470/510 nm for GFP) assists in this process, but only surface tumor nodules are detectable. Additionally, fluorescence from blood contamination, which is visible under the same parameters as GFP, may lead to false identification of possible metastatic lesions.
Sectioning of the lung followed by H&#38;E staining to visualize lung metastasis is a useful method to evaluate micrometastases and other microscopic processes including immune cell infiltration but often requires use of the entire lung tissue for paraffin embedding, sectioning, and staining procedures. Therefore, downstream procedures are not ideal following this method. Although quantifiable, this procedure requires the investigator to evaluate a large number of stained lung sections per animal to ensure that the analysis accounts for the entire 3D structure of the lung. Consequently, this type of examination is time consuming, can lead to counting error, and analysis relies heavily on investigator discretion.
Several in vivo imaging techniques (e.g. MRI, PET, SPECT) are currently used to perform or test biological processes in experimental rodent models 8. In vivo bioluminescent imaging is a common method used to acquire a gross view of metastasis 9. This technique is generally applied to evaluate the presence of luciferase reporter activity due to the accumulation of tumor cells, which are engineered to contain a luciferase response element, that reside in specific organs like the mammary gland after tumor cell implantation and the lung upon spontaneous metastasis 10. Visualization of luciferase reporter activity is induced by the presence of luciferin substrate (D-luciferin). Luciferase catalyzes the oxidative decarboxylation of D-luciferin to oxyluciferin generating bioluminescence. While informative, this method is limited by several factors including substrate stability (i.e. short half-life), adequate distribution of substrate which depends on how it is delivered to experimental animals, and low sensitivity of detection 9. A main merit to this technique is that it is non-invasive, can be performed on live animals, and can lead to the detection of tumor cell metastases in multiple organs that may not have been normally harvested at dissection 9,10.
One positive aspect of in vivo imaging techniques is that the lung tissue is undisturbed allowing for secondary procedures like paraffin embedding or as presented here, QRT-PCR analysis. However, because QRT-PCR is theoretically a more sensitive measure of detection, gross evaluation may not reveal low numbers of tumor cells present in the lung. While useful, the imaging techniques described above can be substituted or supplemented with the QRT-PCR method currently described. QRT-PCR has the potential to provide a sensitive measure of tumor-derived mRNA within a lung.

<table><tbody><tr><td>2-mercaptoethanol</td><td>Fisher</td><td>BP176-100</td><td></td></tr><tr><td>DNAse</td><td>Thermo Scientific</td><td>EN0521</td><td></td></tr><tr><td>GeneJet RNA Isolation Kit</td><td>Thermo Scientific</td><td>K0732</td><td></td></tr><tr><td>iScript Reverse Transcription Supermix for QRT-PCR</td><td>Bio-Rad</td><td>1708841</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix&amp;nbsp;</td><td>Bio-Rad</td><td>172-5121&amp;nbsp;</td><td></td></tr><tr><td>PrimePCR SYBR Green Assay: ERBB2, Human&amp;nbsp;</td><td>Bio-Rad</td><td>100-25636&amp;nbsp;</td><td></td></tr><tr><td>PrimePCR Template for SYBR Green Assay: ERBB2, Human&amp;nbsp;</td><td>Bio-Rad</td><td>100-25716&amp;nbsp;</td><td></td></tr><tr><td>gapdh Primer Sequence</td><td></td><td></td><td>For-ATGGTGAAGGTCGGTGTGAACG Rev-CGCTCCTGGAAGATGGTGATGG</td></tr></tbody></table>

evaluation of lung metastasis in mouse mammary tumor models by quantitative real-time pcr

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

Beyond the time it takes to perform the initial inoculation of tumor cells into the experimental animal and if performing, primary in vivo tumor analysis, the tissue harvest, RNA isolation, and QRT-PCR analysis is a 1-2 day procedure (Figure 1).
An example of gross analysis is bioluminescent imaging to evaluate tumor cells within the lung tissue. Here, a mammary tumorigenesis experiment was performed to...

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

The overall goal of this procedure is to detect the quantity of a specific MRNA from tumor cells in the lung, as a method for analyzing metastasis. The main advantage of this technique is that it is a quantitative measure of metastasis. To perform a lung tumor dissection, after euthanising a mouse using a lethal dose of isofluorine according to the text protocol, place the mouse on a foam board on its back with the limbs spread, and use dissection pins to secure it to the board.After using ethanol to spray the mouse, use forceps to grasp the lower portion of the animal at the center, and with scissors, cut upwards towards the neck through the skin, being careful not to pierce the peritoneal cavity of the mouse. Now, grasp the peritoneum at the lower end of the animal, and begin cutting upwards toward the base of the neck. Carefully cut along the left and right sides of the ribcage, taking care not to sever any blood vessels that may bleed into the thoracic cavity.Remove the rib bones by cutting to the left and right, through the diaphragm and upper portions of the ribcage. Then, using forceps, grasp the trachea of the mouse and pull forward, and use dissection scissors to sever the trachea before removing the lungs from the mouse. Use PBS to gently wash the lung tissue.Then, after gross evaluation of the lung tissue according to the text protocol, if the lungs are to be analyzed immediately, proceed to RNA isolation. To isolate RNA from tissue, begin by homogenizing the tissue using one of the methods listed in the text protocol. Prepare lysis buffer from an RNA isolation kit, and add 20 microliters of tumor captoethanol to each milliliter of lysis buffer.Resuspend every 30 grams of tissue in 300 microliters of lysis buffer, and with a sonicator set at 30%amplitude, sonicate the tissue for 10 seconds. Add 10 microliters of protinase K to 590 microliters of TE buffer, and add 600 microliters of the buffer to 300 microliters of sonicated tissue. Incubate at room temperature for 10 minutes.Next, centrifuge the samples at greater than or equal to 13, 000 times G for 10 minutes to remove the debris. Then transfer the supernatant to new tubes. Add 450 microliters of ethanol to every 900 microliters of supernatant to precipitate the RNA.Then transfer 700 microliters of the lysate ethanol mix to an RNA column. Spin again at greater than or equal to 13, 000 times G for one minute to bind the RNA to the column. Then discard the liquid waste, and add the remaining supernatant to the column, and spin again.Next, add 350 microliters of wash buffer one to the upper portion of the column, and centrifuge the samples for 30 seconds. Discard the liquid waste and replace the column in the tube. Prepare DNase by mixing five units of DNase one enzyme with five microliters of 10X DNase buffer, and 40 microliters of DNase RNase free water for each sample.Add 50 microliters of DNase mix to each column, and incubate at room temperature for 15 minutes. Then add 350 microliters of wash buffer one to the columns and centrifuge the samples for 30 seconds. After discarding the waste solution, add 600 microliters of wash buffer two, and spin for an additional 30 seconds.Discard the liquid before adding 250 microliters of wash buffer two, and then spin for two minutes. Transfer the column into a new collection tube, and add 50 to 100 microliters of RNase DNase free water to the column. After incubating for one minute, spin for one minute.Re-run the collected eluate through the column to increase the RNA yield. For immediate use, keep the samples on ice. For later use, snap freeze on dry ice or liquid nitrogen, and store at minus 80 degrees Celsius until needed.Using a spectrophotometer, quantify the RNA. Then, in sterile RNase DNase free PCR tubes or plates, use 0.5 to 2 micrograms of total RNA to prepare the reaction mix for first transynthesis, as shown in this table. Mix, and gently centrifuge, and program a standard PCR machine using the program shown here.When the reaction is complete, use sterile, nuclease free water to dilute the finished reaction to the desired volume. To carry out real time PCR, use a positive control C DNA, such as human HER2, to set up a standard curve for each primer set by preparing one to four serial dilutions of the C DNA to generate five standards. Next, calculate the number of total samples, which should include the standard curve and experimental samples.Then, in sterile RNase DNase free tubes, prepare a master mix for each experimental gene of interest, as well as an internal control, such as gapdh. Prepare a test plate containing one microliter of C DNase for each standard or experimental sample. Allocate at least one C DNA sample per well, for each primer probe.Add 19 microliters of mastermix per well, for each primer probe. This bioluminescence experiment demonstrates that mouse lungs treated with ACT1, which targets the gap junction protein Connexin 43, appear negative for luciferase activity, suggesting the absence of tumor cells in the tissue. However, analysis of H&E sections of the lung tissue shows micrometastases that were not illuminated by luciferase, suggesting luciferase imaging is not sensitive enough to detect low numbers of tumor cells.Shown here are the results from a qRT-PCR experiment, using a probe specific for HER2, to detect human HER2 plus JIMT1 tumor cells in lung tissue from cells originally transplanted into the mouse memory fat pad. Although no grossly visible metastases were observed on any of the lungs, in two of the 11 samples, qRT-PCR detected metastases. In this figure, a standard curve analysis was prepared to determine the relative HER2 levels normalized to mouse lung gapdh in a cell number series.The results indicate the qRT-PCR, using the HER2 probe, was sensitive enough to detect HER2 levels in as few as 10 cells. Finally, this regression analysis shows the relative cell number in each lung sample, including the positive and negative controls. Once mastered, this technique can be done in six to eight hours, if performed properly.While attempting this procedure, it's important to remember to use precaution to maintain RNA integrity during RNA isolation and reverse transcriptase reaction. After watching this video, you should have a good understanding of how to detect metastasis in whole lung tissue, by quantitative real time PCR.

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JoVE Journal

Medicine

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An Orthotopic Murine Model of Human Prostate Cancer Metastasis

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Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

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Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

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This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

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Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Learn quantitative real-time PCR to detect and quantify tumor-specific mRNA in mouse lung tissue for analyzing metastasis. This method provides sensitive measurement of cancer progression in mammary tumor models.

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

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