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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

PCR-grade quality DNA and RNA from TRIzol-BEB extraction.(a) The concentration of DNA samples from mouse bones using 3 different protocols was determined by measuring UV absorbance (b) Electrophoresis of DNA extracted from mouse tibiae using different protocols on a 1% (wt/vol) agarose gel in 0.5x TAE running buffer (2.0 μg of DNA loaded in each lane). (c) RNA and DNA extracted with TriZOL-BEB from the same bone sample were run on a denaturing gel, showing no evidence of cross contamination in the samples (d) TriZOL-BEB method allows for extraction of DNA and RNA from bone tissues. Alu qPCR of samples with 103 MDA-231 cells spiked into a mouse tibia (d) with corresponding mouse b-actin expression (e) using up to 200 ng DNA extracted from bone tissue, (n = 3).
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f1: PCR-grade quality DNA and RNA from TRIzol-BEB extraction.(a) The concentration of DNA samples from mouse bones using 3 different protocols was determined by measuring UV absorbance (b) Electrophoresis of DNA extracted from mouse tibiae using different protocols on a 1% (wt/vol) agarose gel in 0.5x TAE running buffer (2.0 μg of DNA loaded in each lane). (c) RNA and DNA extracted with TriZOL-BEB from the same bone sample were run on a denaturing gel, showing no evidence of cross contamination in the samples (d) TriZOL-BEB method allows for extraction of DNA and RNA from bone tissues. Alu qPCR of samples with 103 MDA-231 cells spiked into a mouse tibia (d) with corresponding mouse b-actin expression (e) using up to 200 ng DNA extracted from bone tissue, (n = 3).

Mentions: Several established nucleic acid extraction techniques were systematically evaluated and we found that a modification of the TRIzolTM protocol using a back extraction buffer (TRIzol-BEB method) allowed for the most efficient extraction of DNA from bones. Yields were superior to those obtained with a commercial kit (DNeasy kit, Qiagen) and another commonly-used protocol based on proteinase K digestion (Fig. 1a,b). It is important to note here that RNA expression data and DNA can be extracted from the same sample with the TRIzol-BEB method, while the other techniques yield only genomic information to be quantified. Other methods of DNA extraction from bone, which did not consistently yield serviceable DNA, included using EDTA decalcification, NaOH and boiling, or other TRIzol modifications recommended by the manufacturer (data not shown). For TRIzol-BEB DNA extraction, frozen samples were pulverized using a carbon steel mortar and pestle, previously cooled in liquid nitrogen. By keeping the surface of the pestle between -100 and 0 °C, we were able to exploit the Leidenfrost effect, which slows the evaporation time of liquid nitrogen and allows for nucleation of the suspended sample powder on the collection tool, thereby significantly reducing contamination and sample loss. Powdered samples were then resuspended in TRIzol and thoroughly vortexed before proceeding with extraction and precipitation. The range of RNA yield was 0.23–1.24 μg per mg of starting tissue, (x̄ = 0.63 μg/mg of tissue). Cross-contamination was not detectable in the RNA or DNA, which were extracted from the same sample, as shown by gel electrophoresis (Fig. 1c).


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)

PCR-grade quality DNA and RNA from TRIzol-BEB extraction.(a) The concentration of DNA samples from mouse bones using 3 different protocols was determined by measuring UV absorbance (b) Electrophoresis of DNA extracted from mouse tibiae using different protocols on a 1% (wt/vol) agarose gel in 0.5x TAE running buffer (2.0 μg of DNA loaded in each lane). (c) RNA and DNA extracted with TriZOL-BEB from the same bone sample were run on a denaturing gel, showing no evidence of cross contamination in the samples (d) TriZOL-BEB method allows for extraction of DNA and RNA from bone tissues. Alu qPCR of samples with 103 MDA-231 cells spiked into a mouse tibia (d) with corresponding mouse b-actin expression (e) using up to 200 ng DNA extracted from bone tissue, (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: PCR-grade quality DNA and RNA from TRIzol-BEB extraction.(a) The concentration of DNA samples from mouse bones using 3 different protocols was determined by measuring UV absorbance (b) Electrophoresis of DNA extracted from mouse tibiae using different protocols on a 1% (wt/vol) agarose gel in 0.5x TAE running buffer (2.0 μg of DNA loaded in each lane). (c) RNA and DNA extracted with TriZOL-BEB from the same bone sample were run on a denaturing gel, showing no evidence of cross contamination in the samples (d) TriZOL-BEB method allows for extraction of DNA and RNA from bone tissues. Alu qPCR of samples with 103 MDA-231 cells spiked into a mouse tibia (d) with corresponding mouse b-actin expression (e) using up to 200 ng DNA extracted from bone tissue, (n = 3).
Mentions: Several established nucleic acid extraction techniques were systematically evaluated and we found that a modification of the TRIzolTM protocol using a back extraction buffer (TRIzol-BEB method) allowed for the most efficient extraction of DNA from bones. Yields were superior to those obtained with a commercial kit (DNeasy kit, Qiagen) and another commonly-used protocol based on proteinase K digestion (Fig. 1a,b). It is important to note here that RNA expression data and DNA can be extracted from the same sample with the TRIzol-BEB method, while the other techniques yield only genomic information to be quantified. Other methods of DNA extraction from bone, which did not consistently yield serviceable DNA, included using EDTA decalcification, NaOH and boiling, or other TRIzol modifications recommended by the manufacturer (data not shown). For TRIzol-BEB DNA extraction, frozen samples were pulverized using a carbon steel mortar and pestle, previously cooled in liquid nitrogen. By keeping the surface of the pestle between -100 and 0 °C, we were able to exploit the Leidenfrost effect, which slows the evaporation time of liquid nitrogen and allows for nucleation of the suspended sample powder on the collection tool, thereby significantly reducing contamination and sample loss. Powdered samples were then resuspended in TRIzol and thoroughly vortexed before proceeding with extraction and precipitation. The range of RNA yield was 0.23–1.24 μg per mg of starting tissue, (x̄ = 0.63 μg/mg of tissue). Cross-contamination was not detectable in the RNA or DNA, which were extracted from the same sample, as shown by gel electrophoresis (Fig. 1c).

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