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TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton.

Preston Campbell J, Mulcrone P, Masood SK, Karolak M, Merkel A, Hebron K, Zijlstra A, Sterling J, Elefteriou F - Sci Rep (2015)

Bottom Line: Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity.We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification.This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization in xenograft models.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America [2] Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity. We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification. This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization in xenograft models.

No MeSH data available.


Related in: MedlinePlus

Alu PCR is a sensitive technique for detecting xenograft cells within the bone.Correlation of ct of Alu with number of human MDA-MB-231 cells spiked into murine BMSC (a) and whole mouse femora (b). (c) Cell number could be quantified by Alu qPCR from low numbers of MDA-MB-231GFP sorted into entire mouse humerii (). Comparison of sensitivity of FACS with Alu qPCR (d) in detecting MDA-MB-231GFP cells spiked into 106 mouse BMSCs. Dashed lines are placed at the level of background signal from negative controls (no human cells) using the gating for eGFP shown in (e), n = 3.
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f2: Alu PCR is a sensitive technique for detecting xenograft cells within the bone.Correlation of ct of Alu with number of human MDA-MB-231 cells spiked into murine BMSC (a) and whole mouse femora (b). (c) Cell number could be quantified by Alu qPCR from low numbers of MDA-MB-231GFP sorted into entire mouse humerii (). Comparison of sensitivity of FACS with Alu qPCR (d) in detecting MDA-MB-231GFP cells spiked into 106 mouse BMSCs. Dashed lines are placed at the level of background signal from negative controls (no human cells) using the gating for eGFP shown in (e), n = 3.

Mentions: Though qPCR of primate Alu repeats has been used routinely in forensics and anthropology, attempts at using Alu qPCR to quantify human tumor cells in xenograft models have been hampered by extraction difficulties and background signal from human DNA contamination. Genomic DNA extracted using our modified TRIzol-BEB protocol was used for Alu qPCR and gave accurate linear quantification of 101 to 106 human cell numbers from serial dilutions of MDA-MB-231 cells spiked into 106 murine bone marrow cells (Fig. 2a). Accurate cell number counts were also seen when Alu qPCR was used to count MDA-MB-231 cells that were spiked directly into entire pulverized mouse femora (Fig. 2b), demonstrating that the combined methods of TRIzol-BEB extraction and Alu qPCR provide a sensitive method to quantify disseminated human metastatic cells in bone tissue.


TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton.

Preston Campbell J, Mulcrone P, Masood SK, Karolak M, Merkel A, Hebron K, Zijlstra A, Sterling J, Elefteriou F - Sci Rep (2015)

Alu PCR is a sensitive technique for detecting xenograft cells within the bone.Correlation of ct of Alu with number of human MDA-MB-231 cells spiked into murine BMSC (a) and whole mouse femora (b). (c) Cell number could be quantified by Alu qPCR from low numbers of MDA-MB-231GFP sorted into entire mouse humerii (). Comparison of sensitivity of FACS with Alu qPCR (d) in detecting MDA-MB-231GFP cells spiked into 106 mouse BMSCs. Dashed lines are placed at the level of background signal from negative controls (no human cells) using the gating for eGFP shown in (e), n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4536516&req=5

f2: Alu PCR is a sensitive technique for detecting xenograft cells within the bone.Correlation of ct of Alu with number of human MDA-MB-231 cells spiked into murine BMSC (a) and whole mouse femora (b). (c) Cell number could be quantified by Alu qPCR from low numbers of MDA-MB-231GFP sorted into entire mouse humerii (). Comparison of sensitivity of FACS with Alu qPCR (d) in detecting MDA-MB-231GFP cells spiked into 106 mouse BMSCs. Dashed lines are placed at the level of background signal from negative controls (no human cells) using the gating for eGFP shown in (e), n = 3.
Mentions: Though qPCR of primate Alu repeats has been used routinely in forensics and anthropology, attempts at using Alu qPCR to quantify human tumor cells in xenograft models have been hampered by extraction difficulties and background signal from human DNA contamination. Genomic DNA extracted using our modified TRIzol-BEB protocol was used for Alu qPCR and gave accurate linear quantification of 101 to 106 human cell numbers from serial dilutions of MDA-MB-231 cells spiked into 106 murine bone marrow cells (Fig. 2a). Accurate cell number counts were also seen when Alu qPCR was used to count MDA-MB-231 cells that were spiked directly into entire pulverized mouse femora (Fig. 2b), demonstrating that the combined methods of TRIzol-BEB extraction and Alu qPCR provide a sensitive method to quantify disseminated human metastatic cells in bone tissue.

Bottom Line: Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity.We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification.This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization in xenograft models.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America [2] Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity. We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification. This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization in xenograft models.

No MeSH data available.


Related in: MedlinePlus