Name: droplet digital trap (ddtrap): adaptation of the telomere repeat amplification protocol to droplet digital polymerase chain reaction
Uploaded: 2019-05-03T15:00:00
Description: Watch this Scientific Journal Video about Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction at JoVE.com

The authors would like to acknowledge funding sources from the National Institutes of Health (NIH) (NCI-R00-CA197672-01A1). Small cell lung cancer lines (SHP77 and H82) were a generous gift from Drs. John Minna and Adi Gazdar from the UT Southwestern Medical Center.

1. Buffer preparation and storage
<ol>
	<li>Prepare 50 mL of 1x stock RNase-/DNase-free NP-40 lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% [vol/vol] NP-40, 10% [vol/vol] glycerol, 150 mM NaCl, 5 mM &#946;-mercaptoethanol, and 0.1 mM 4-benzenesulfonyl fluoride hydrochloride (AEBSF)). This buffer can be aliquoted and stored at -20 &#176;C for future use. Avoid freeze/thaw cycles in order to obtain an optimal lysis of cells.</li>
	<li>Prepare 50 mL of 10x st...

<ol>
	<li>Frenck, R. W., Blackburn, E. H., Shannon, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rate+of+telomere+sequence+loss+in+human+leukocytes+varies+with+age.">The rate of telomere sequence loss in human leukocytes varies with age.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (10), 5607-5610 (1998).</li><li>Morin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+telomere+terminal+transferase+enzyme+is+a+ribonucleoprotein+that+synthesizes+TTAGGG+repeats.">The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats.</a> Cell. 59 (3), 521-529 (1989).</li><li>de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+telomeres+solve+the+end-protection+problem.">How telomeres solve the end-protection problem.</a> Science. 326 (5955), 948-952 (2009).</li><li>Shay, J. W., Wright, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+telomeres+and+telomerase+in+cancer.">Role of telomeres and telomerase in cancer.</a> Seminars in Cancer Biology. 21 (6), 349-353 (2011).</li><li>Kim, W., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+telomere+regulation+of+gene+expression:+Telomere+looping+and+telomere+position+effect+over+long+distances+(TPE-OLD).">Long-range telomere regulation of gene expression: Telomere looping and telomere position effect over long distances (TPE-OLD).</a> Differentiation. 99, 1-9 (2018).</li><li>Robin, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+position+effect:+regulation+of+gene+expression+with+progressive+telomere+shortening+over+long+distances.">Telomere position effect: regulation of gene expression with progressive telomere shortening over long distances.</a> Genes Development. 28 (22), 2464-2476 (2014).</li><li>Shay, J. W., Wright, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence+and+immortalization:+role+of+telomeres+and+telomerase.">Senescence and immortalization: role of telomeres and telomerase.</a> Carcinogenesis. 26 (5), 867-874 (2005).</li><li>Norton, J. C., Holt, S. E., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection+of+human+telomerase+activity.">Enhanced detection of human telomerase activity.</a> DNA Cell Biology. 17 (3), 217-219 (1998).</li><li>Herbert, B. S., Hochreiter, A. E., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonradioactive+detection+of+telomerase+activity+using+the+telomeric+repeat+amplification+protocol.">Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol.</a> Nature Protocols. 1 (3), 1583-1590 (2006).</li><li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (16), 9236-9241 (1999).</li><li>Ludlow, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+telomerase+enzyme+activity+determination+using+droplet+digital+PCR+with+single+cell+resolution.">Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.</a> Nucleic Acids Research. 42 (13), e104 (2014).</li><li>Huang, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Maintenance+of+Telomere+Length+in+CD28++T+Cells+During+T+Lymphocyte+Stimulation.">The Maintenance of Telomere Length in CD28+ T Cells During T Lymphocyte Stimulation.</a> Scientific Reports. 7 (1), 6785 (2017).</li><li>Ludlow, A. T., Shelton, D., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ddTRAP:+A+Method+for+Sensitive+and+Precise+Quantification+of+Telomerase+Activity.">ddTRAP: A Method for Sensitive and Precise Quantification of Telomerase Activity.</a> Methods Molecular Biology. 1768, 513-529 (2018).</li></ol>

The measurement of telomerase activity is critical to a plethora of research topics including, but not limited to, cancer, telomere biology, aging, regenerative medicine, and structure-based drug design. Telomerase RNPs are low abundant, even in cancer cells, making the detection and study of this enzyme challenging. In this paper, we described the step-by-step procedures for the newly developed ddTRAP assay to robustly quantify telomerase activity in cells. By combining the traditional telomerase extension reaction with...

Telomeres are dynamic DNA-protein complexes at the ends of linear chromosomes. Human telomeres are composed of an array of 5&#39;-TTAGGGn hexameric repeats which vary in length between 12&#8211;15 kilobases (kb) at birth1. Human telomerase, the ribonucleoprotein enzyme that maintains the telomeres, was first identified in HeLa cell lysates (cancer cell line)2. Together, telomeres and telomerase play a major role in a spectrum of biological processes such as genome protection, gene regulation, and cancer cell immortality3,4,5,6.
Human telomerase is comprised primarily of two key components, namely telomerase reverse transcriptase and telomerase RNA (hTERT and hTERC, respectively). The protein subunit, hTERT, is the catalytically active reverse transcriptase component of the telomerase enzyme. The RNA template, hTERC, provides telomerase with the template to extend and/or maintain telomeres. Most human somatic tissues have no detectable telomerase activity. The inability of DNA polymerase to extend the end of the lagging strand of DNA along with the lack of telomerase leads to the progressive shortening of telomeres after every round of cellular division. These phenomena lead to telomere shortening in most somatic cells until they reach a critically shortened length, whereby cells enter a state of replicative senescence. The maximal number of times a cell can divide is dictated by its telomere length and this block to continued cell division is thought to prevent progression to oncogenesis7. Cancer cells are able to overcome telomere-induced replicative senescence and continue to proliferate by utilizing telomerase to maintain their telomeres. Approximately 90% of cancers activate telomerase, making telomerase activity critically important in both cancer detection and treatment.
The development of the TRAP assay in the 1990s was instrumental in the identification of the necessary components of the telomerase enzyme, as well as for the measurement of telomerase in a wide range of cells and tissues, both normal and cancerous. The original gel-based PCR assay used radioactively labeled DNA substrates to detect telomerase activity. In 2006, the assay was adapted into a nonradioactive form using fluorescently labeled substrates8,9. By using fluorescently labeled substrates, users were able to visualize the telomerase extension products as bands on a gel by exposing it to the correct excitation wavelength. The sensitivity of the TRAP assay and its ability to detect telomerase activity in crude cell lysates has made this assay the most widely used method for telomerase activity detection. However, the TRAP assay has limitations. The assay is gel-based, making it difficult to perform the necessary replicates in moderate to high-throughput studies, and thus, proper statistical analysis is rarely achieved. Furthermore, the gel-based assay is difficult to quantify reliably due to the inability of detecting less than twofold differences in telomerase activity between samples. Overcoming these two limitations is critical for enzymatic activity assays such as the TRAP to move to clinical or industry settings for the detection of telomerase activity in patient samples or drug design studies.
Digital PCR was initially developed in 1999 as a means to convert the exponential and analog nature of PCR into a linear and digital assay10. Droplet digital PCR (ddPCR) is the most recent innovation of the original digital PCR methodology. Droplet digital PCR came about with the advent of advanced microfluidics and oil-in-water emulsion chemistry to reliably generate stable and equally sized droplets. Unlike gel-based and even quantitative PCR (qPCR), ddPCR generates absolute quantification of the input material. The key to ddPCR is the generation of ~20,000 individual reactions by partitioning samples into droplets. Following end-point PCR, the droplet reader scans each droplet in a flow-cytometer-like fashion, counting, sizing, and recording the presence or absence of fluorescence in each individual droplet (i.e., absence or presence of PCR amplicons in each droplet). Then, using Poisson&#8217;s distribution, input molecules are estimated based on the ratio of positive droplets to the total number of droplets. This number represents an estimate of the number of input molecules in each PCR. Furthermore, ddPCR is performed and analyzed on a 96-well plate which allows the user to run many samples, as well as perform biological and technical replicates for proper statistical analysis. As a result, we have combined the powerful quantification and moderate-throughput nature of ddPCR with the TRAP assay to develop the ddTRAP assay11. This assay is designed for users to study and robustly quantify absolute telomerase activity from biological samples11,12. The sensitivity of the ddTRAP allows the quantification of telomerase activity from limited and precious samples, including single-cell measurements. Furthermore, users can also study the effects of telomerase manipulations and/or drugs with absolute quantification of less than twofold changes (~50% differences). The ddTRAP is the natural evolution of the TRAP assay into the digital and higher-throughput nature of modern laboratory experiments and clinical settings.

<table>
	<tbody>
		<tr>
			<td>1 M Tris-HCl pH 8.0</td>
			<td>Ambion</td>
			<td>AM9855G</td>
			<td>RNAse/DNAse free</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>Ambion</td>
			<td>AM9530G</td>
			<td>RnAse/DNAse free</td>
		</tr>
		<tr>
			<td>0.5 M EDTA pH 8.0</td>
			<td>Ambion</td>
			<td>AM9261</td>
			<td>RNAse/DNAse free</td>
		</tr>
		<tr>
			<td>Surfact- Amps NP-40</td>
			<td>Thermo Scientific</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ultrapure Glycerol</td>
			<td>Invitrogen</td>
			<td>15514011</td>
			<td>RNAse/DNAse free</td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>516732</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease Free H20</td>
			<td>Ambion</td>
			<td>AM9932</td>
			<td>RNAse/DNAse free</td>
		</tr>
		<tr>
			<td>2.5 mM dNTP mix</td>
			<td>Thermo Scientific</td>
			<td>R72501</td>
			<td>2.5 mM of each dATP, dCTP, dGTP and dTTP</td>
		</tr>
		<tr>
			<td>2 M KCl</td>
			<td>Ambion</td>
			<td>AM9640G</td>
			<td>RNAse/DNAse free</td>
		</tr>
		<tr>
			<td>100% Tween-20</td>
			<td>Fisher</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EGTA pH 8.0</td>
			<td>Fisher</td>
			<td>50-255-956</td>
			<td>RNAse/DNAse free</td>
		</tr>
		<tr>
			<td>Telomerase Substrate (TS) Primer</td>
			<td>Integrated DNA Technology (IDT)</td>
			<td>Custom Primer (HPLC Purified)</td>
			<td>5&#39;- AATCCGTCGAGCAGAGTT-3&#39;</td>
		</tr>
		<tr>
			<td>ACX (Revers) Primer</td>
			<td>Integrated DNA Technology (IDT)</td>
			<td>Custom Primer (HPLC Purified)</td>
			<td>5&#39;- GCGCGGCTTACCCTTACCCTTACCCTAACC -3&#39;</td>
		</tr>
		<tr>
			<td>Thin walled (250 ul) PCR grade tubes</td>
			<td>USA Scientific</td>
			<td>1402-2900</td>
			<td>strips, plates, tubes etc.</td>
		</tr>
		<tr>
			<td>QX200 ddPCR EvaGreen Supermix</td>
			<td>Bio Rad</td>
			<td>1864034</td>
			<td></td>
		</tr>
		<tr>
			<td>Twin-Tec 96 Well Plate</td>
			<td>Fisher</td>
			<td>Eppendorf 951020362</td>
			<td></td>
		</tr>
		<tr>
			<td>Piercable foil heat seal</td>
			<td>Bio Rad</td>
			<td>1814040</td>
			<td></td>
		</tr>
		<tr>
			<td>Droplet generator cartidges (DG8)</td>
			<td>Bio Rad</td>
			<td>1863008</td>
			<td></td>
		</tr>
		<tr>
			<td>Droplet generator oil</td>
			<td>Bio Rad</td>
			<td>1863005</td>
			<td></td>
		</tr>
		<tr>
			<td>Droplet generator gasket</td>
			<td>Bio Rad</td>
			<td>1863009</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Thermocycler T100</td>
			<td>Bio Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer</td>
			<td>Bio Rad</td>
			<td>1814000</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader and Quantasoft Software</td>
			<td>Bio Rad</td>
			<td>1864001 and 1864003</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Droplet Reader Oil</td>
			<td>Bio Rad</td>
			<td>1863004</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease Free Filtered Pipette Tips</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>10 ul, 20 ul , 200 ul and 1000 ul</td>
		</tr>
	</tbody>
</table>

droplet digital trap (ddtrap): adaptation of the telomere repeat amplification protocol to droplet digital polymerase chain reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Using the ddTRAP, telomerase activity was measured in a cell panel consisting of the following cell lines (Figure 1): nonsmall cell lung cancer (H2882, H1299, Calu6, H920, A549, and H2887), small cell lung cancer (H82 and SHP77), and telomerase-negative fibroblasts (BJ). One million cell pellets were lysed in NP-40 buffer, and telomerase extension reactions were performed in biological triplicates. A common and highly recommended negative control is the &#822...

Watch this Scientific Journal Video about Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction at JoVE.com

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

The ddTRAP assay provides the ability to obtain a sensitive, robust, and highly quantitative measurement of telomerase enzymatic activity in cells. The ability to run up to 96 samples per run in a medium-throughput fashion offers us a great advantage. We can run the controls and replicates for proper statistical analysis on the data collected.Immediately prior to cell lysing, thaw a frozen aliquot of NP-40 lysis buffer. Once thawed, place the lysis buffer on ice. Add PMSF protease inhibitor to the NP-40 lysis buffer to reach a final concentration of 0.2 millimolar.Then, add 40 microliters of the lysis buffer to the tube containing a cell pellet to reach a cell equivalence of 25, 000 cells per microliter. Gently pipette the lysate up and down in order to break open the cells. Try to avoid making bubbles.Allow the cells to lyse on ice for 30 minutes. Gently vortex the lysate every 10 minutes to prevent clusters of cell debris from forming. During the cell lysis, prepare a master mix in a microcentrifuge tube for the telomerase extension reaction, and store on ice.After the cell lysis, pipette 48 microliters of extension master mix into each PCR tube. Add two microliters of diluted lysate to each PCR tube to reach a final cell equivalence of 50 cells per microliter, and continue the telomerase extension reaction according to the manuscript. Prepare a master mix in a microcentrifuge tube for the ddTRAP, and keep it at room temperature.Pipette 19.8 microliters of ddPCR master mix into each PCR tube. Add 2.2 microliters of the previous extension reaction solution to each tube, leaving a total volume of 22 microliters. To set up the droplet generation cartridge, first load 20 microliters of the prepared solution into the middle sample well in the cartridge.Gently tap the side of the cartridge to move any bubbles to the top of the solution. All wells of the cartridge must be loaded with sample prior to loading oil. Then, load 70 microliters of droplet generation oil into the left oil well.Secure the gasket in place by tethering it to the ends of the cartridge, and place the loaded and assembled cartridge into the droplet generator. The generator shows a solid green light to inform the cartridge is placed properly. After 60 to 90 seconds, when the generator light stops blinking, the cycle is complete.Now remove the cartridge. Gently remove the gasket from the cartridge. Use a multichannel pipette to pipette approximately 40 microliters of the newly generated droplets from the right well into a 96-well PCR plate.Heat seal the plate with aluminum foil PCR plate seals once all the samples are loaded in the 96-well plate to prevent evaporation. Then, load the 96-well plate into a thermocycler to perform the PCR reaction. To begin, load the 96-well plate into the droplet reader.Orient the plate properly so that the A1 sample matches that of the holder. Open the software associated with the droplet reader. Double-click the first well, A01, to open the sample well editor screen.Click Experiment, and select the type of assay to be used from the drop-down menu. Select QX200 ddPCR EvaGreen Supermix as the correct detection method. Click Apply in the lower right-hand side of the well editor screen to save the user-defined settings to all of the highlighted wells.Next, click on Target in the well editor screen, and click the Target drop-down menu to select either unknown, reference, or no-template control to define the type of sample. In the Sample Name section, label all the samples and click Apply. Click Run to read the plate.Select either Columns or Rows in the Run Options screen when prompted to inform about the orientation the plate should be read in. Determine the number of accepted droplets for each sample by double-clicking on the individual wells or column and row headers to provide a table of information. For the ddTRAP, samples with 10, 000 or more accepted droplets are valid for further analysis.Highlight the wells representing sample replicates and NTC samples. Manually set the threshold for the samples by clicking on the icon for setting thresholds on the bottom left of the screen. In this protocol, telomerase activity was measured in a cell panel consisting of nine cell lines of lung cancer and telomerase-negative fibroblasts.A threshold was set at around 2, 000 fluorescence amplitude for all three biological replicates for SHP77, H2887, and NTC. Positive droplets had a fluorescence intensity around 6, 000 fluorescence amplitude, which formed a clear population at the top, and separate from negative droplets around 1, 100 fluorescence amplitude. The total telomerase extension products per cell equivalent between all the samples were estimated from the measured concentration of the nucleic acids, which represents the telomerase activity in lung cancer lines.Any RNase contamination during the lysis or extension reaction steps will destroy telomerase enzymatic activity since it is a ribonucleoprotein complex. ddTRAP can be followed up with ddPCR on hTERT mRNA expression levels in the cell. The two data sets allows us to correlate expression of hTERT with telomerase activity and dig deeper into potential telomerase regulatory mechanisms such as alternative splicing.We can explore manipulations to specific splicing factors and their effects on telomerase activity. Currently, we are designing oligonucleotide splicing modulators as potential drugs targeting telomerase activity.

Research

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