Name: telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (trap) assay
Uploaded: 2014-09-02T13:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay at JoVE.com

This work was partially supported by B.G. Negev technology and Linda Powers's contribution.

Animal experiments were approved by the ethical committee for animal experiments in the Ben-Gurion University (IL-39-08-2010).
1. Mouse Brain Protein Extraction
<ol>
	<li>Prepare 3 tubes of 15 ml each containing 5 ml Ringer&#39;s solution and place tubes on ice.</li>
	<li>Sacrifice the mouse using Isoflurane anesthesia (CAUTION &#8211; use chemical hood) for 10-20 sec followed by cervical dislocation and immediate decapitation.</li>
	<li>Remove the brain from the skull and rinse for a few seconds with ice cold Ringer&#39;s solution to prevent damage to the brain tissue.</li>
	<li>Using a surgical blade, separate the cerebellum (CR) and brainstem (BS) from the frontal cortex. Next, separate the cerebellum from the brainstem and cut the frontal lobe (FL) from the frontal cortex; remove the olfactory bulb from the frontal lobe. Place the 3 brain regions in the designated 15 ml tubes.</li>
	<li>Homogenize the tissue in each tube. Add fresh ice cold Ringer&#39;s solution so each tube contains the same volume and centrifuge for 7 min at 800 x g.</li>
	<li>Remove the supernatant and add 100 &#181;l of CHAPS lysis buffer to each tube. Mix well, by passing the solution several times (~10) through a 1 ml syringe with a 21 G needle, and incubate on ice for 30 min.</li>
	<li>Transfer the contents of each tube to a new micro-centrifuge tube and centrifuge for 30 min at 13,000 x g. Transfer the supernatant to a new micro-centrifuge tube and determine the protein concentration using a previously established method. Store the protein extracts in aliquots of 10 &#181;l at -80 &#176;C.</li>
</ol>
2. TRAP Assay Preparation of Instruments, Reaction Solutions, and Protein Dilution Calculations
<ol>
	<li>Prepare appropriate number of labeled micro-centrifuge tubes for protein extract dilutions and telomerase reaction buffer. Perform all pipetting in the protocol using filter tips to prevent contamination.</li>
	<li>Thaw the following solutions on ice: TRAP reaction mix 10x, TS primer, dNTP&#39;s, UPW and protein extracts (see table of materials for reagents and reagents concentrations).</li>
	<li>Dilute the solution of the protein extract to a final concentration of 1 &#181;g/&#181;l using ultra-pure water (UPW).</li>
</ol>
3. Telomerase Reaction
<ol>
	<li>Prepare telomerase reaction mix by adding the following to a micro-centrifuge tube: 2 &#181;l TRAP mix (10x), 1 &#181;l TS primer (0.1 &#181;g), 1 &#181;l dNTPs (10 mM) and 15 &#181;l UPW. For multiple samples, multiply aforementioned volumes by the number of samples, plus 1.</li>
	<li>Add 19 &#181;l of the reaction mix to each reaction tube and add 1 &#181;l of the diluted protein extract (1 &#181;g proteins) to each tube for a final volume of 20 &#181;l.</li>
	<li>Place the reaction tubes in a 30 &#176;C pre-heated water bath for 45 min.</li>
</ol>
4. PCR Reaction
<ol>
	<li>Fifteen min before the end of the telomerase reaction, thaw the following solutions: ACX primer, Titanium Taq Buffer, UPW.</li>
	<li>Prepare PCR reaction mix by adding the following to a micro-centrifuge tube: 2.5 &#181;l Titanium Taq Buffer (10x), 1 &#181;l ACX primer (0.1 &#181;g), 2 &#181;l UPW (1 &#956;l when IC is used) and 0.5 &#181;l Titanium Taq Polymerase (50x). For multiple samples, multiply aforementioned volumes by the number of samples plus 2.</li>
	<li>Add 6 &#181;l of the PCR mix to each PCR tube and add the entire volume of the telomerase reaction (20 &#181;l) to each tube for a final volume of 26 &#181;l.</li>
	<li>Insert the PCR tubes to a PCR thermo cycler and run the following program: 
	- 90 &#176;C &#8211; 2 min. 
	- 94 &#176;C &#8211; 30 sec.* 
	- 60 &#176;C &#8211; 30 sec.* 
	- 72 &#176;C &#8211; 45 sec.* 
	- 72 &#176;C &#8211; 2 min. 
	Store PCR products at -20 &#176;C. 
	* = 34 cycles</li>
</ol>
5. Agarose Mini-gel Preparation, Samples Running, and Gel Staining
<ol>
	<li>Add 25 ml chilled electrophoresis buffer (TBE) and a stir bar to a beaker that is 2-4 times the volume of the buffer solution and slowly sprinkle in 1.125 g of the high-resolution (HR) agarose powder while the solution is rapidly stirred. Remove the stir bar, cover the beaker with a plastic wrap and pierce in a small hole for ventilation. Heat the beaker in the microwave on &#34;Low&#34; power for 3 min and then switch to &#34;High&#34; power and heat the solution in short pulses with care not to cause spilling. Note that when the foam created by heating becomes large bubbles and disappears after a few seconds, the agarose is ready.</li>
	<li>Cast the gel to a horizontal mini-gel apparatus, after the gel solidifies at room temperature allow the gel to chill for 20 min at 4 &#176;C before use.</li>
	<li>Load each lane with 25 &#181;l of TRAP sample (20 &#181;l TRAP PCR products and 5 &#181;l 6x gel-loading dye) and run at 110 V for 3 hr in a 4 &#176;C cold room.</li>
	<li>Prepare nucleic acid stain 3x working solution by adding 15 &#181;l 10,000x nucleic-acid stain, stock solution to 50 ml Double Distilled Water (DDW) with 0.1 M NaCl (0.29 g). Stain the gel for 20 min with gentle shaking.</li>
	<li>Use UV transilluminator with 302 nm wavelength to view the TRAP ladder DNA bands.</li>
</ol>

<ol>
	<li>Urquidi, V., Tarin, D., Goodison, S. <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+telomerase+in+cell+senescence+and+oncogenesis.">Role of telomerase in cell senescence and oncogenesis.</a> Annu Rev Med. 51, 65-79 (2000).</li><li>Cong, Y., 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=Actions+of+human+telomerase+beyond+telomeres.">Actions of human telomerase beyond telomeres.</a> Cell Res. 18, 725-732 (2008).</li><li>Tichon, A., 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=Oxidative+stress+protection+by+novel+telomerase+activators+in+mesenchymal+stem+cells+derived+from+healthy+and+diseased+individuals.">Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals.</a> Curr.Mol. Med. 13, 1010-1022 (2013).</li><li>Lannilli, F., Zalfa, F., Gartner, A., Bagni, C., Dotti, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+TERT+associates+to+RNA+granules+in+fully+mature+neurons:+Role+in+the+translational+control+of+the+cell+cycle+inhibitor+p15INK4B.">Cytoplasmic TERT associates to RNA granules in fully mature neurons: Role in the translational control of the cell cycle inhibitor p15INK4B.</a> PloS one. 8, e66602 (2013).</li><li>De Jesus, B. B., Blasco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+telomerase+activation+in+extending+health+span+and+logevity.">Potential of telomerase activation in extending health span and logevity.</a> Curr opinion in Cell Biology. 24, 1-5 (2012).</li><li>Blackburn, E., Collins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase:+An+RNP+enzyme+synthesizes+DNA.">Telomerase: An RNP enzyme synthesizes DNA.</a> Cold Spring Harb Perspect Biol. 3, 1-9 (2011).</li><li>Klapper, W., Shin, T., Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+telomerase+activity+and+TERT+expression+during+brain+development+in+mice.">Differential regulation of telomerase activity and TERT expression during brain development in mice.</a> J. Neurosci. Res. 64, 252-260 (2001).</li><li>Caporaso, G. L., Lim, D. A., Alvarez-Buylla, A., Chao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+activity+in+the+subventricular+zone+of+adult+mice.">Telomerase activity in the subventricular zone of adult mice.</a> Mol. Cell Neurosci. 23, 693-702 (2003).</li><li>Eitan, 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=Novel+Telomerase-increasing+compound+in+mouse+brain+delays+the+onset+of+amyotrophic+lateral+sclerosis.">Novel Telomerase-increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis.</a> EMBO Mol. Med. 4, 313-329 (2012).</li><li>Kim, N. W., Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+quantification+and+characterization+of+telomerase+activity+by+the+telomeric+repat+amplification+protocol+(TRAP).">Advances in quantification and characterization of telomerase activity by the telomeric repat amplification protocol (TRAP).</a> Nuc. Acid Res. 25, 2595-2597 (1997).</li></ol>

The authors declare that they have no competing financial interests.

Analysis of telomerase activity in the mouse brain via the TRAP assay consists of 4 major steps: 1) proteins extraction from specific regions of the brain tissue 2) TS primer elongation by telomerase - telomerase reaction 3) amplification of the telomerase reaction products by PCR 4) separation of the PCR products using high resolution agarose gel.
This modified TRAP assay addresses two major difficulties that rise from the traditional assay: the use of radioactive dCTP&#39;s for sensitive det...

Telomerase is a ribonucleoprotein composed of telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, and a RNA component (TERC). The canonical role of telomerase is to preserve the proper length of the telomeres by adding repetitive sequences (TTAGGG) to the telomeric ends, therefore promoting genomic integrity, and cell proliferation1. Additional roles were attributed to TERT in cells, i.e. it confers resistance to apoptosis induced by various damaging agent2, protects human mesenchymal stem cells from oxidative stress3, and plays a pro-survival role in fully differentiated neurons by its association to TIA1 positive RNA granules4.
TERT is expressed and active mainly in somatic dividing cells and in most types of cancers, while the enzyme expression and activity levels are tightly regulated in non-dividing cells5,6. Studies have shown that in the rodent brain, telomerase activity becomes undetectable by postnatal day 10, while TERT mRNA is maintained at lower levels into adulthood7. Other studies demonstrated telomerase activity within the adult sub-ventricular zone, the olfactory bulb, the hippocampus, and in the adult cerebellum and cortex8. We recently demonstrated that novel compounds that increase telomerase expression and activity in the brain and spinal cords exerted neuroprotective effects in N-Methyl-D-aspartate (NMDA) - injected mice, delayed the onset and progression of the amyotrophic lateral sclerosis disease in SOD1 transgenic mice and increased the survival of motor neurons in the spinal cord of these mice9. To fully understand the pro-survival role of telomerase in neurons and in the brain, it is essential to develop a simple and relatively sensitive fast assay for the detection of telomerase activity in the various regions of the brain.
The telomeric repeat amplification protocol (TRAP) is a well-known sensitive assay combining the canonical activity of telomerase and the polymerase chain reaction (PCR). In this assay, telomerase adds TTAGGG repeats to a telomerase substrate (TS primer) oligonucleotide, followed by PCR amplification which generates 6 base-pairs (bp) DNA ladder using the TS reverse primer &#8211; ACX. 32P labeled dCTP&#8217;s are used to detect the low amount of PCR products. The DNA ladder is resolved by sequencing polyacrylamide gel electrophoresis (PAGE). Both the intensity of the DNA bands and the length of the DNA ladder reflect the amount of active enzyme molecules and their processivity respectively10.
This traditional assay holds some major difficulties: The danger of exposure to radioactive substances, large and hard to handle sequencing PAGE and the time consumption of the gel running and film exposure of the radioactive product (overnight in both cases).
This protocol offers an improved non-radioactive agarose mini-gel based assay. The DNA 6 bp ladder is resolved using 4.5% high-resolution agarose gel and the detection is achieved using highly sensitive nucleic acid stain. The combination of using high resolution agarose mini-gel and sensitive nucleic acid stain offers easy to handle assay, eliminates the danger of exposure to radioactivity and significantly shorten the overall assay duration from 3 days to several hours.

<table><tbody><tr><td>TRAP Mix (10x)</td><td></td><td></td><td>Made manually, mix the following in UPW: 630 mM KCl, 200 mM Tris-HCl pH 8.2, 10 mM EDTA, 15 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 1 mg/ml BSA, 0.5% TWEEN (purchased from Sigma). Aliquots are frozen at -20 &amp;deg;C.</td></tr><tr><td>Ringer&#039;s solution</td><td></td><td></td><td>Made manually, mix the following in DDW: 124 mM NaCl, 3 mM KCl, 1.25 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O, 2 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 26 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 1.802 g/L D-(+)-glucose anhydrous.</td></tr><tr><td>Isoflurane</td><td>Minrad INC.</td><td>NDC 60307-110-10</td><td>Handle with care in a chemical hood</td></tr><tr><td>dNTPs (10 mM)</td><td>Sigma</td><td>D7295</td><td></td></tr><tr><td>TS primer (5&#039;-AATCCGTCGAGCAGAGTT-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>ACX primer (5&#039;-GCGCGGCTTACCCTTACCCTTACCCTAACC-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>Titanium Taq Polymerase/Buffer</td><td>Clontech</td><td>S1792/S1793</td><td></td></tr><tr><td>High Resolution agarose</td><td>Sigma</td><td>A4718</td><td>Any high quality high-resolution agarose may be sutible</td></tr><tr><td>Mini-gel apparatus</td><td>Bio-Rad</td><td>170-4466</td><td></td></tr><tr><td>Nucleic Acid Stain (GEL-RED)</td><td>Biotium</td><td>41003</td><td></td></tr><tr><td>IC primer (5&#039;-ATCGCTTCTCGGCCTTTT-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>CHAPS lysis buffer</td><td>Cell Signaling Technology</td><td>9852S</td><td>Add PMSF (protease inhibitor) to a final concentration of 1 mM</td></tr><tr><td>Ultra Pure Water (UPW)</td><td>Biological Industries</td><td>01-866-1B</td><td></td></tr><tr><td>CD-1 mice</td><td>HARLAN Laboratories INC.</td><td></td><td></td></tr></tbody></table>

telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (trap) assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Whole cell protein extracts were prepared from the frontal lobe (FL), brain stem (BS) and cerebellum (CR), derived from 3 CD-1 female and 3 CD-1 male mice at the ages of 1 and 3 months old for the modified TRAP assay and 3 CD-1 male mice at the age of 2 months old for the radioactive TRAP assay. Telomerase activity was detected in 1 &#181;g proteins extract using the modified TRAP assay and 2 &#181;g proteins extract for the radioactive TRAP assay. Detection of telomerase activity by the radioactive TRAP assay and by the...

Watch this Scientific Journal Video about Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay at JoVE.com

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

telomerase-activity-various-regions-mouse-brain-non-radioactive

Research

JoVE Journal

Neuroscience

14.6K Views.  Ben-Gurion University of the Negev. Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Video: Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Genetics

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

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.

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.

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

Cancer Research

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

Medicine

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

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

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence