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Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.

Ludlow AT, Robin JD, Sayed M, Litterst CM, Shelton DN, Shay JW, Wright WE - Nucleic Acids Res. (2014)

Bottom Line: The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes.Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR.The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390-9039, USA.

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ddTRAP detection of mouse telomerase. Extracts from one million HeLa and one million NIH3T3 mouse fibroblasts were prepared. Following lysis (40 μl), 1-μl of the lysate (25 000 cell/μl) was added to a 50 μl extension reaction (extension product concentration = 500 cells/μl). One-microliter of the extension reaction was used for either the gel based TRAP or the ddTRAP assay. (A) Gel analysis (10% PAGE) of HeLa compared to NIH3T3 cells. Gel was stained with Gel red, a double stranded DNA binding dye. (B) ddTRAP total products generated (molecules per microliter output multiplied by 20 μl). Quantification showed an eight-fold difference in extension products Eqs. = equivalents. (C) Quantification of scanned images from A (ImageJ, http://imagej.nih.gov/ij/, (22)) showing a seven-fold difference which is similar to ddTRAP quantification.
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f7: ddTRAP detection of mouse telomerase. Extracts from one million HeLa and one million NIH3T3 mouse fibroblasts were prepared. Following lysis (40 μl), 1-μl of the lysate (25 000 cell/μl) was added to a 50 μl extension reaction (extension product concentration = 500 cells/μl). One-microliter of the extension reaction was used for either the gel based TRAP or the ddTRAP assay. (A) Gel analysis (10% PAGE) of HeLa compared to NIH3T3 cells. Gel was stained with Gel red, a double stranded DNA binding dye. (B) ddTRAP total products generated (molecules per microliter output multiplied by 20 μl). Quantification showed an eight-fold difference in extension products Eqs. = equivalents. (C) Quantification of scanned images from A (ImageJ, http://imagej.nih.gov/ij/, (22)) showing a seven-fold difference which is similar to ddTRAP quantification.

Mentions: There are sequence differences in the telomerase RNA component between human and mouse TERC (26). We examined whether ddTRAP, similar to gel based TRAP, was able to detect mouse telomerase activity using immortal NIH3T3 mouse fibroblasts (Figure 7). The magnitude of the difference of telomerase activity between TRAP and ddTRAP was similar in HeLa compared to NIH3T3 cells (Figure 7). Optimization of telomerase substrates that are more compatible with the mouse telomerase RNA component will be needed for determination of absolute molecule counts of mouse telomerase activity. The present data does not address how efficiently mouse telomerase works on the TS primer, but ddTRAP offers a suitable quantitative surrogate to traditional gel-based methods.


Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.

Ludlow AT, Robin JD, Sayed M, Litterst CM, Shelton DN, Shay JW, Wright WE - Nucleic Acids Res. (2014)

ddTRAP detection of mouse telomerase. Extracts from one million HeLa and one million NIH3T3 mouse fibroblasts were prepared. Following lysis (40 μl), 1-μl of the lysate (25 000 cell/μl) was added to a 50 μl extension reaction (extension product concentration = 500 cells/μl). One-microliter of the extension reaction was used for either the gel based TRAP or the ddTRAP assay. (A) Gel analysis (10% PAGE) of HeLa compared to NIH3T3 cells. Gel was stained with Gel red, a double stranded DNA binding dye. (B) ddTRAP total products generated (molecules per microliter output multiplied by 20 μl). Quantification showed an eight-fold difference in extension products Eqs. = equivalents. (C) Quantification of scanned images from A (ImageJ, http://imagej.nih.gov/ij/, (22)) showing a seven-fold difference which is similar to ddTRAP quantification.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4117742&req=5

f7: ddTRAP detection of mouse telomerase. Extracts from one million HeLa and one million NIH3T3 mouse fibroblasts were prepared. Following lysis (40 μl), 1-μl of the lysate (25 000 cell/μl) was added to a 50 μl extension reaction (extension product concentration = 500 cells/μl). One-microliter of the extension reaction was used for either the gel based TRAP or the ddTRAP assay. (A) Gel analysis (10% PAGE) of HeLa compared to NIH3T3 cells. Gel was stained with Gel red, a double stranded DNA binding dye. (B) ddTRAP total products generated (molecules per microliter output multiplied by 20 μl). Quantification showed an eight-fold difference in extension products Eqs. = equivalents. (C) Quantification of scanned images from A (ImageJ, http://imagej.nih.gov/ij/, (22)) showing a seven-fold difference which is similar to ddTRAP quantification.
Mentions: There are sequence differences in the telomerase RNA component between human and mouse TERC (26). We examined whether ddTRAP, similar to gel based TRAP, was able to detect mouse telomerase activity using immortal NIH3T3 mouse fibroblasts (Figure 7). The magnitude of the difference of telomerase activity between TRAP and ddTRAP was similar in HeLa compared to NIH3T3 cells (Figure 7). Optimization of telomerase substrates that are more compatible with the mouse telomerase RNA component will be needed for determination of absolute molecule counts of mouse telomerase activity. The present data does not address how efficiently mouse telomerase works on the TS primer, but ddTRAP offers a suitable quantitative surrogate to traditional gel-based methods.

Bottom Line: The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes.Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR.The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390-9039, USA.

Show MeSH
Related in: MedlinePlus