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 detection of the PCR products and PAGE separation of the DNA ladder. High resolution agarose gel simplifies the detection of the telomerase reaction products (the DNA ladder) compared to the hard to handle sequencing PAGE. In addition, the nucleic acid staining is a non-toxic solution with high sensitivity needed for the detection of small amount of DNA.
Special attention should be given to the following critical steps: 1) keeping the time between tissue removal and homogenization to a minimum to preserve protein integrity 2) CHAPS lysis buffer refreezing should be avoided 3) protein extracts should be kept in aliquots and thawed once before use. Avoid refreezing of samples.
Problems may occur during the gel preparation step since high concentration agarose gel is prone to spillover and requires close attention during cooking. The technique holds several limitations with the necessity of running the gel in a cold room. In addition, the low amount of PCR products cannot be viewed on a UV table.
Telomerase activity level varies among different tissues and is considered to be low in the brain. This protocol offers a more sensitive detection method, compare to the traditional radioactive assay enabling better detection of telomerase activity in the brain. Although different time-efficient methods are available (one step TRAP assay kits, using a labeled TS primer for detection) these methods are significantly more expensive. Moreover, the use of agarose eliminates the toxic hazard of using polyacrylamide gel.
As revealed from the representative results, by using the above described method, it is possible to demonstrate the differences in telomerase activity levels between the various regions of the brain and between male and female. This method can be used for the detection of telomerase activity in other normal tissues with low telomerase activity.

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 modified TRAP assay shows that a better visualization and resolution of the telomerase DNA products is observed with the modified assay as seen by the length and intensity of the DNA ladder (Figure 1A vs. 1C and 1D). A gradual reduction in the telomerase DNA products is observed when decreasing concentrations of protein extracts, derived from glioblastoma U-251 cell line, was used suggesting the sensitivity of the modified TRAP assay method for the detection of telomerase activity at lower concentrations of proteins extract (Figure 1B).
A significantly higher telomerase activity is seen in the FL and BS regions compared to the CR in 3 month old females and males as revealed by the intensity of the DNA bands and the length of the DNA products ladder. In addition, it is shown that during the mouse transition into adulthood telomerase activity increases in the FL and decreases in the BS and CR (Figure 1D). Comparison between the sexes demonstrates that in the FL telomerase activity is higher in females in contrast to the BS and CR showing higher telomerase activity in males.
In tissues expressing low levels of telomerase activity we have found that the detection of the DNA ladder using PCR is more efficient without using the internal control (IC) primer, since the IC utilizes the TS as a forward primer thus interfering with the DNA ladder amplification. The reproducibility of the data is achieved by repeating each experiment several times.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51865/51865fig1highres.jpg" width="500" /> 
Figure 1. Telomerase activity in the mouse brain: Traditional versus modified TRAP assay. A) Telomerase activity in 3 brain regions analyzed by radioactive TRAP assay (2 months old CD-1 male mice, 2 &#181;g proteins extract). B) Sensitivity of the modified TRAP assay is shown by decreasing concentrations of the proteins extract derived from glioblastoma U-251 cell line. C, D) Telomerase activity in 3 brain regions of 1 (C) and 3 (D) months old CD-1 female and male mice. The DNA ladder starts at the 50 bp band with 6 bp increments. (NC - negative control, IC- internal control, BS &#8211; brain stem, CR &#8211; cerebellum, FL &#8211; frontal lobe).

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

Nanyang Technological University

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

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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

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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 ...

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Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

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Biochemistry

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

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

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their alterations are perhaps associated with a pro-inflammatory state. The TgF344-AD rat strain has been designed to express human APP and human PS1&#916;E9 genes, encoding for amyloid proteins A&#946;-40 and A&#946;-42 and displays amyloid pathology and cognitive deficits with aging. The TgF344-AD rat model is used in this study to evaluate the cellular origin of the 18 kDa translocator protein (TSPO, a marker of glial cell activation) binding, and the 5HT2A-receptor (5HT2AR) serotonin receptor levels that are possibly disrupted in AD. The technique presented here is Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT), a quantitative cell-type-specific technique complementary to in vivo PET or SPECT or ex vivo/in vitro autoradiography techniques. It quantifies the same radiolabeled tracer used prior for imaging, using a &#947; counter after cytometry cell sorting. This allows determining the cellular origin of the radiolabeled protein with high cellular specificity and sensitivity. For example, studies with FACS-RTT showed that (i) the increase in TSPO binding was associated with microglia in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation, (ii) an increase in TSPO binding at 12- and 18-months was associated with astrocytes first, and then microglia in the TgF344-AD rats compared to wild type (WT) rats, and (iii) the striatal density of 5HT2AR decreases in astrocytes at 18 months in the same rat AD model. Interestingly, this technique can be extended to virtually all radiotracers.

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT2A-receptor expression in Alzheimer&#39;s Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model.

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

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

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Chapters in this video

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

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

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

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

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

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

Cell Type-specific Gene Expression Profiling in the Mouse Liver