This work was supported in part by the Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT) (15cm0106102h0002, K.M.) and the Project for Cancer Research and Therapeutic Evolution (P-CREATE) (16cm01061150001, K.M.) from Japan Agency for Medical Research and Development, AMED; the Takeda Science Foundation (Y.M.); Grant of the Princess Takamatsu Cancer Research Fund (13-24520, Y.M.); and JSPS KAKENHI Grant Number JP16K07133 (Y.M.).

1. Reagent Setup
<ol>
	<li>Reagents used for Cell Synchronization
	<ol>
		<li>To generate Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 2.5 mM thymidine, dissolve 30.28 mg of thymidine per 1 mL of tissue culture grade water to prepare 125 mM thymidine. Filtrate the solution with a 0.22 &#956;m syringe filter unit. Add 1 mL of freshly prepared 125 mM thymidine per 50 mL of DMEM supplemented with 10% (vol/vol) fetal bovine serum (FBS).</li>
		<li>To generate DMEM containing 0.1 &#956;g/mL of nocodazole, dissolve nocodazole in dimethyl sulfoxide (DMSO) to prepare 5 &#956;g/&#956;L nocodazole. Add 1 &#181;L of 5 &#181;g/ &#181;L nocodazole per 50 mL of DMEM supplemented with 10% (vol/vol) FBS.</li>
		<li>To generate DMEM containing 0.002% (vol/vol) of DMSO, add 1 &#181;L of DMSO per 50 mL of DMEM supplemented with 10% (vol/vol) FBS.</li>
	</ol>
	</li>
	<li>Reagents used for IP-RdRP assay
	<ol>
		<li>Prepare lysis buffer A: 150 mM NaCl, 20 mM Tris-HCl (pH 7.4) and 0.5% (vol/vol) Nonidet P-40 (NP-40). Store the reagent at 4 &#176;C.</li>
		<li>Prepare 5x acetate buffer: 50 mM 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)-KOH (pH 7.8), 500 mM potassium acetate and 20 mM MgCl2. Store the reagent at room temperature.</li>
		<li>Prepare Wash buffer 1: 1x acetate buffer, 10% (vol/vol) glycerol, 0.1% (vol/vol) Triton X-100 and 0.06x protease inhibitor cocktail, ethylenediamenetetraacetic acid (EDTA)-free. Prepare Wash buffer 1 on the day of use or the day before use. Store the reagent at 4 &#176;C.</li>
		<li>Prepare AGC solution: 1x acetate buffer, 10% (vol/vol) glycerol and 0.02% (wt/vol) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonat (CHAPS). Store the reagent at 4 &#176;C.</li>
		<li>To generate AGC solution containing 2 mM CaCl2, add 100 &#181;L of 1 M CaCl2 per 50 mL of AGC solution. Store the reagent at 4 &#176;C.</li>
		<li>To generate AGC solution containing 3 mM ethylene glycol bis (b-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA), add 375 &#181;L of 0.4 M EGTA (pH 7.5) per 50 mL of AGC solution. Store the reagent at 4 &#176;C.</li>
		<li>Prepare Wash buffer 2: 1x acetate buffer and 0.02% (wt/vol) CHAPS. Store the reagent at 4 &#176;C.</li>
		<li>Prepare HMD solution: 0.2 M HEPES-KOH (pH 7.8), 40 mM MgCl2 and 2 mM dithiothreitol (DTT). Store the reagent at -20 &#176;C. 
		NOTE: HMD solution should be renewed at least in three months.</li>
		<li>Prepare 2x Proteinase K buffer: 20 mM Tris-HCl (pH 7.6), 20 mM EDTA and 1% (wt/vol) sodium dodecyl sulfate (SDS). Store the reagent at -20 &#176;C.</li>
		<li>Prepare 2x loading buffer: 95% (vol/vol) formamide, 20 mM EDTA and 1% (wt/vol) Orange G. Store the reagent at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of HeLa cells
NOTE: Prepare HeLa cells synchronized in mitotic phase (manipulated) or dividing asynchronously (unmanipulated). Culture the cells in the presence of 5% CO2 at 37 &#176;C.
<ol>
	<li>Synchronization of HeLa cells in mitosis
	<ol>
		<li>Plate 1 x 106 of HeLa cells with 10 mL of DMEM supplemented with 10% (vol/vol) FBS in a 10 cm culture dish.</li>
		<li>Two days after plating, replace the medium with 10 mL of DMEM containing 2.5 mM thymidine and 10% (vol/vol) FBS. Culture the cells for 24 h.</li>
		<li>Wash the cells three times with 10 mL of phosphate buffered saline (PBS) (-), and culture the cells with 10 mL of DMEM containing 10% (vol/vol) FBS for 6 h.</li>
		<li>Replace the medium with 10 mL of DMEM containing 0.1 &#181;g/mL of nocodazole and 10% (vol/vol) FBS, and culture the cells for 14 h.</li>
		<li>Shake the culture dish gently, and retrieve the supernatant containing the detached mitotic cells. Count the cell number for the IP-RdRP assay.</li>
		<li>Centrifuge the sample at 490 x g for 5 min at 4 &#176;C, and discard the supernatant. 
		NOTE: The cell pellet can be stored at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of unmanipulated HeLa cells
	<ol>
		<li>Plate 1 x 106 of HeLa cells with 10 mL of DMEM supplemented with 10% (vol/vol) FBS in a 10 cm culture dish.</li>
		<li>Two days after plating, replace the medium with 10 mL of DMEM containing 10% (vol/vol) FBS. Culture the cells for 30 h.</li>
		<li>Replace the medium with 10 mL of DMEM containing 0.002% (vol/vol) DMSO and 10% (vol/vol) FBS, and culture the cells for 14 h.</li>
		<li>Collect the cells by trypsinization using 0.7 mL of trypsin (2.5 g/L) containing 1 mM EDTA. Count the cell number for the IP-RdRP assay.</li>
		<li>Centrifuge the sample at 490 x g for 5 min at 4 &#176;C, and discard the supernatant. 
		NOTE: The cell pellet can be stored at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. IP-RdRP Assay
<ol>
	<li>Protein A-agarose bead preparation
	<ol>
		<li>Transfer 1 mL (bed volume 500 &#181;L) of affinity resin to a 1.5 mL microcentrifuge tube. Centrifuge at 800 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Add 800 &#181;L of Lysis buffer A to the beads, and mix well by inverting the tube several times. Centrifuge at 800 x g for 3 min at 4 &#176;C, and discard the supernatant. Repeat this step twice (total three washes).</li>
		<li>Add 800 &#181;L of Lysis buffer A to the beads, mix well by inverting the tube several times. Rotate the tube overnight (or for at least 4 h) at 4 &#176;C on a rotary shaker.</li>
		<li>Centrifuge the tube at 800 x g for 3 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Add 500 &#181;L of Lysis buffer A to the beads, and mix well by inverting the tube several times. Store the beads at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of cell lysate
	<ol>
		<li>(Optional) If the cells are frozen in step 2, place and thaw the frozen cells on ice until the cells become loose.</li>
		<li>Wash the cells once with 10 mL of PBS (-). Centrifuge the sample at 1,450 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Lyse the cells with ice-cold Lysis buffer A (1 mL of Lysis buffer A per 1 x 107 of the cells) by gentle pipetting.</li>
		<li>Sonicate the sample in a 1.5 mL tube with following conditions; set amplification of the sonicator at 25%, and sonicate for 10 s.</li>
		<li>Centrifuge the sample at 20,400 x g for 15 min at 4 &#176;C.</li>
		<li>Collect the supernatant. Transfer 1 mL each of the supernatant into 1.5 mL microcentrifuge tubes. 
		CAUTION: Perform all procedures under RNase-free conditions.</li>
	</ol>
	</li>
	<li>Immunoprecipitation
	<ol>
		<li>Pre-clear the lysate from step 3.2.6 by adding 40 &#181;L (bead volume 20 &#181;L) of prewashed protein A-agarose beads per 1 mL of the lysate. Mix well by inverting the tube several times and rotate the sample for 30 min at 4 &#176;C.</li>
		<li>Centrifuge the sample at 13,000 x g for 1 min at 4 &#176;C, and recover the supernatant.</li>
		<li>Add 40 &#181;L (bed volume 20 &#181;L) of prewashed protein A-agarose beads and 10 &#181;g of anti-human TERT antibody (clone: 10E9-2)8 into the pre-cleared lysate. Mix well by inverting the tube several times and rotate the sample at 4 &#176;C over night.</li>
		<li>Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant.</li>
		<li>Wash the beads with Wash buffer 1: add 1 mL of Wash buffer 1 to the beads, mix well by inverting the tube, and rotate the sample for 5 min at 4 &#176;C. Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant. Repeat this step three additional times.</li>
		<li>Wash the beads once with AGC solution containing 2 mM CaCl2: add 1 mL of AGC solution containing 2 mM CaCl2 to the beads, mix well by inverting the tube, and rotate the sample for 5 min at 4 &#176;C. Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant.</li>
		<li>Treat the beads with Micrococcal nuclease (MNase): prepare the MNase reaction mixture described in Table 1. Resuspend the beads in 60 &#181;L of the MNase reaction mixture by gentle pipetting. Shake the sample gently (20 to 25 rpm) on a turntable of a shaker set in a Peltier-type incubator for 15 min at 25 &#176;C. 
		NOTE: It is very important to use an undulating shaker and strictly follow the speed limit in this step. Other type of shakers, such as rotating mixers, are not recommended.</li>
		<li>Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant.</li>
		<li>Wash the beads twice with AGC solution containing 3 mM EGTA: add 1 mL of AGC solution containing 3 mM EGTA to the beads, mix well by inverting the tube, and rotate the sample for 5 min at 4 &#176;C. Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant. Repeat this step once.</li>
		<li>Wash the beads once with Wash buffer 2: add 1 mL of Wash buffer 2 to the beads, mix well by inverting the tube, and rotate the sample for 5 min at 4 &#176;C. Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant. Keep the beads on ice while setting up RdRP reaction. 
		CAUTION: Perform all procedures under RNase-free conditions.</li>
	</ol>
	</li>
	<li>RdRP reaction
	<ol>
		<li>Prepare a RdRP reaction mixture in a new tube as described in Table 2.</li>
		<li>Add 6 &#181;L of [&#945;-32P]UTP (3,000 Ci/mmol) to the mixture and mix well by pipetting. 
		NOTE: [&#945;-32P]ATP, [&#945;-32P]CTP, or [&#945;-32P]GTP can be used for the reaction instead of [&#945;-32P]UTP.</li>
		<li>Add 1 &#181;L of template RNA (1 &#181;g/&#181;L) to the mixture and mix well by pipetting. 
		NOTE: To establish the RdRP assay, the following RNA template is recommended: 5&#39;-GGGAUCAUGUGGGUCCUAUUACAUUUUAAACCCA-3&#39; 9.</li>
		<li>Resuspend the beads with 20 &#181;L of the RdRP reaction mixture from step 3.4.3 by pipetting.</li>
		<li>Shake the sample gently (20 to 25 rpm) on a turntable of a shaker set in a peltier-type incubator for 2 h at 32 &#176;C.</li>
		<li>Add 5.4 &#181;L of Proteinase K (20 mg/mL) and 45.4 &#181;L of 2x Proteinase K buffer to the reaction mixture. Mix by pipetting and shake (20 to 25 rpm) the sample on a turntable set in a Peltier-type incubator for 30 min at 37 &#176;C.</li>
		<li>Add 109.2 &#181;L of RNase-free water to the sample.</li>
		<li>Add 200 &#181;L of acid phenol-chloroform to the sample. Mix well by vortex, and then centrifuge the sample at 21,100 x g for 5 min at room temperature. Transfer the aqueous phase to a new tube. Repeat this step once.</li>
		<li>Add 20 &#181;L of 3 M sodium acetate (pH = 5.2), 4 &#181;L of precipitation carrier and 250 &#181;L of ethanol to the aqueous phase. Shake the tube well for mixing, then centrifuge the sample at 21,100 x g for 20 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Wash the pellet with 300 &#181;L of cold 70% (vol/vol) ethanol stored at -20 &#176;C. Centrifuge the sample at 21,100 x g for 15 min at 4 &#176;C.</li>
		<li>Discard the supernatant and air-dry the pellet. 
		CAUTION: Perform all procedures under RNase-free conditions. 
		NOTE: The pellet from step 3.4.11 can be stored at -20 &#176;C for at least 2 days.</li>
	</ol>
	</li>
	<li>RNase treatment
	<ol>
		<li>Resuspend the pellet from step 3.4.11 in 20 &#181;L of RNase-free water.</li>
		<li>Prepare a RNase reaction mixture in a new tube as described in Table 3.</li>
		<li>Add 180 &#181;L of the RNase reaction mixture to the sample, and incubate the tube for 2 h at 37 &#176;C.</li>
		<li>Add 2.3 &#181;L of 10% (vol/vol) SDS to the sample and incubate for 15 min at 37 &#176;C.</li>
		<li>Add 2 &#181;L of Proteinase K (20 mg/mL) to the sample and incubate for 15 min at 37 &#176;C.</li>
		<li>Add 205 &#181;L of acid phenol-chloroform to the sample. Mix well by vortex, then centrifuge the sample at 21,100 x g for 5 min at room temperature. Transfer the aqueous phase to a new tube.</li>
		<li>Add 20 &#181;L of 3 M sodium acetate (pH 5.2), 4 &#181;L of precipitation carrier and 250 &#181;L of ethanol to the aqueous phase. Shake the tube well for mixing, then centrifuge the sample at 21,100 x g for 20 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Wash the pellet with 300 &#181;L of cold 70% (vol/vol) ethanol stored at -20 &#176;C. Centrifuge the sample at 21,100 x g for 15 min at 4 &#176;C.</li>
		<li>Discard the supernatant and air-dry the pellet. 
		NOTE: The pellet from step 3.5.9 can be stored at -20 &#176;C for at least 2 days.</li>
	</ol>
	</li>
	<li>Gel electrophoresis and autoradiography
	<ol>
		<li>Resuspend the sample with 20 &#181;L of RNase-free water.</li>
		<li>Add 20 &#181;L of 2x loading buffer.</li>
		<li>Boil the sample for 10 min at 95 &#176;C. Crush the sample on the ice.</li>
		<li>Run the gel. In case the template size is 34 nts (see also step 3.4.3), 7 M Urea-10% polyacrylamide gel is used for denaturing gel electrophoresis.</li>
		<li>Dry the gel with a gel dryer.</li>
		<li>Expose the film to the dried-gel within an X-ray cassette with an intensifying screen at -80 &#176;C.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Martinez, P., 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=Telomeric+and+extra-telomeric+roles+for+telomerase+and+the+telomere-binding+proteins.">Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins.</a> Nat Rev Cancer. 11 (3), 161-176 (2011).</li><li>Nakamura, T. M., 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=Telomerase+catalytic+subunit+homologs+from+fission+yeast+and+human.">Telomerase catalytic subunit homologs from fission yeast and human.</a> Science. 277 (5328), 955-959 (1997).</li><li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT.">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455 (7213), 633-637 (2008).</li><li>Salgado, P. S., 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+structure+of+an+RNAi+polymerase+links+RNA+silencing+and+transcription.">The structure of an RNAi polymerase links RNA silencing and transcription.</a> PLoS Biol. 4 (12), e434 (2006).</li><li>Castel, S. E., Martienssen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+the+nucleus:+roles+for+small+RNAs+in+transcription,+epigenetics+and+beyond.">RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond.</a> Nat Rev Genet. 14 (2), 100-112 (2013).</li><li>Sugiyama, T., Cam, H., Verdel, A., Moazed, D., Grewal, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-dependent+RNA+polymerase+is+an+essential+component+of+a+self-enforcing+loop+coupling+heterochromatin+assembly+to+siRNA+production.">RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production.</a> Proc Natl Acad Sci U S A. 102 (1), 152-157 (2005).</li><li>Maida, Y., 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=An+RNA-dependent+RNA+polymerase+formed+by+TERT+and+the+RMRP+RNA.">An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA.</a> Nature. 461 (7261), 230-235 (2009).</li><li>Maida, Y., 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=Involvement+of+telomerase+reverse+transcriptase+in+heterochromatin+maintenance.">Involvement of telomerase reverse transcriptase in heterochromatin maintenance.</a> Mol Cell Biol. 34 (9), 1576-1593 (2014).</li><li>Maida, Y., Yasukawa, M., Masutomi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+RNA+Synthesis+by+RNA-Dependent+RNA+Polymerase+Activity+of+Telomerase+Reverse+Transcriptase.">De Novo RNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase.</a> Mol Cell Biol. 36 (8), 1248-1259 (2016).</li><li>Okamoto, N., 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=Maintenance+of+tumor+initiating+cells+of+defined+genetic+composition+by+nucleostemin.">Maintenance of tumor initiating cells of defined genetic composition by nucleostemin.</a> Proc Natl Acad Sci U S A. 108 (51), 20388-20393 (2011).</li><li>van Dijk, A. A., Makeyev, E. V., Bamford, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+viral+RNA-dependent+RNA+polymerization.">Initiation of viral RNA-dependent RNA polymerization.</a> J Gen Virol. 85 (Pt 5), 1077-1093 (2004).</li></ol>

The authors have nothing to disclose.

The IP-RdRP assay is a sensitive method to detect RdRP activity of human TERT. TERT protein is highly expressed in mitotic HeLa cells, in which TERT forms the RdRP complex8,9,10. This suggests that mitotic HeLa cells are an optimal material to detect RdRP activity. In the protocol described above, mitotic and non-synchronized HeLa cells are included as a positive and a negative example, respectively. As shown in Figure 1A, RdRP assay products from the recommended RNA template in mitotic HeLa cells show broad radioactive signals between 20 to 30 nt. In addition to HeLa cells, we have performed the assay with different types of cell lines, and found that the signal pattern can be changed in different cell types9: some show a strong signal only around 30 nt, some show a similar pattern with HeLa cells. We have also performed the assay with RNA templates other than the 34 nt template8, short and long (~300 nt) RNAs with various sequences, and successfully obtained RdRP products from those templates although there may be some preference. For the first trial, however, we recommend using HeLa cells in mitotic phase and the 34 nt RNA template. RdRPs can generate double-stranded RNA both in a primer-dependent and in a primer-independent manner11. We have reported that TERT preserves this property as human RdRP7,9; TERT synthesizes dsRNA from an RNA template with 3&#39;-foldback structure through a back-priming mechanism7. For the 34 nt RNA template, TERT synthesizes complementary strands without using primers9. To specifically detect RdRP products synthesized in a primer-independent manner, i.e. de novo synthesized RNA products, [&#945;-32P]NTP can be replaced with [&#947;-32P]NTP in the RdRP reaction9.
MNase treatment of TERT immune complexes on beads is a critical step to achieve desired results. If the MNase treatment is performed too long or with intensive shaking, the RdRP products would be reduced remarkably. To avoid such a trouble, we strongly recommend to strictly follow the protocol carefully. HMD solution is another critical factor for success. If you find that the signals are very weak, replace the HMD solution to a newly prepared one.
Throughout the protocol, one should take great care to avoid RNase contamination. Ribonuclease treatment should be performed with dedicated equipment. After manipulating the Ribonuclease, one should discard tips and tubes with RNase immediately, eliminate RNase with specialized solution (e.g. RNase Quiet), and change groves.
TERT interacts with not only TERC but also many kinds of endogenous RNAs, and we have reported part of them7. Modification in the IP-RdRP assay, such as the IP-RdRP assay without MNase treatment, will provide a full list of endogenous RNA templates for TERT-associated RdRP activity and bioinformatic guide on their sequence features. We have successfully applied this protocol to tissue lysate. Because TERT is expressed in a variety of normal and tumor tissues, this assay might be useful to investigate non-canonical enzymatic function of TERT in human.

Human telomerase reverse transcriptase (TERT) is well known as the catalytic subunit of telomerase, and it elongates telomere using telomerase RNA component (TERC), the specific RNA template1. Although TERT polymerizes telomeric DNA as a component of telomerase, the structural and phylogenetic analyses indicate that TERT is closely related with viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase, and shares domains with these polymerases2,3,4. RdRP is the enzyme that generates complementary RNA strand to a template RNA. The enzyme is encoded not only in viruses but also in model organisms, such as plant, yeast and worm, and double-stranded RNA synthesis by RdRP contributes to transcriptional and post-transcriptional gene silencing in these organisms5,6. Although human RdRP had been missing for a long time, we found RdRP activity in human TERT in 20097.
We first confirmed RdRP activity of TERT with recombinant protein7, then established a sensitive in vitro assay to detect RdRP activity of endogenous TERT8. Here, we demonstrate the in vitro RdRP assay (IP-RdRP assay) for endogenous TERT. This method starts with immunoprecipitation (IP) of endogenous TERT, and is followed by in vitro RdRP reaction, in which radioactive ribonucleotides are incorporated into nascent RNA strands.

<table border="0" cellpadding="0" cellspacing="0" width="1082">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:464px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:155px;">Wako</td>
			<td style="width:119px;">044-29765</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum (FBS)</td>
			<td>CORNING</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin mixed solution</td>
			<td>nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-Ethylenediamenetetraacetic acid (EDTA) solution</td>
			<td>nacalai tesque</td>
			<td>32777-44</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline (PBS(-))</td>
			<td>Wako</td>
			<td>166-23555</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thymidine</td>
			<td>nacalai tesque</td>
			<td>07147-61</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nocodazole</td>
			<td>Sigma</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>nacalai tesque</td>
			<td>08904-85</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>nacalai tesque</td>
			<td>31333-45</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris(hydroxymethyl)aminomethane</td>
			<td>nacalai tesque</td>
			<td>35434-21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>nacalai tesque</td>
			<td>18321-05</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nonidet P-40 (NP-40)</td>
			<td>nacalai tesque</td>
			<td>25223-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Protein A Plus Agarose</td>
			<td>Thermo scientific</td>
			<td align="right">22812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)</td>
			<td>nacalai tesque</td>
			<td>17514-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium hydroxide</td>
			<td>Wako</td>
			<td>168-21815</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium acetate</td>
			<td>nacalai tesque</td>
			<td>28405-05</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate (MgCl2&#8226;6H2O)</td>
			<td>Wako</td>
			<td>135-00165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>nacalai tesque</td>
			<td>17045-65</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyoxyethylene(10) octylphenyl ether (Triton X-100)</td>
			<td>Wako</td>
			<td>169-21105</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete, EDTA-free</td>
			<td>Roche Applied Science</td>
			<td>11 873 580 001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate (CaCl2&#8226;2H2O)</td>
			<td>nacalai tesque</td>
			<td>06731-05</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micrococcal nuclease (MNase)</td>
			<td>Takara Bio</td>
			<td>2910A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylene glycol bis (b-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>nacalai tesque</td>
			<td>15214-92</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonat (CHAPS)</td>
			<td>nacalai tesque</td>
			<td>07957-64</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td>nacalai tesque</td>
			<td>14128-91</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rCTP, rATP, rUTP, rGTP, 100mM each</td>
			<td>Promega</td>
			<td>E6000</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">[a-32P]UTP (3,000 Ci/mmol)</td>
			<td>PerkinElmer</td>
			<td>NEG507H</td>
			<td>Use fresh RI for the assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase inhibitor</td>
			<td>TOYOBO</td>
			<td>SIN-201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td>Takara</td>
			<td align="right">9033</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td>Life Technologies</td>
			<td>AM9720</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3 M Sodium acetate</td>
			<td>NIPPON GENE</td>
			<td>316-90081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol (99.5)</td>
			<td>nacalai tesque</td>
			<td>14713-95</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dr. GenTLE Precipitation Carrier</td>
			<td>Takara Bio</td>
			<td align="right">9094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase One Ribonuclease</td>
			<td>Promega</td>
			<td>M4265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate (SDS)</td>
			<td>nacalai tesque</td>
			<td>02873-75</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formamide, deionized</td>
			<td>nacalai tesque</td>
			<td>16345-65</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate (EDTA)</td>
			<td>nacalai tesque</td>
			<td>15130-95</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orange G</td>
			<td>nacalai tesque</td>
			<td>25401-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2 incubator</td>
			<td>e.g., ASTEC</td>
			<td>SCA-325DRS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>e.g., TOMY</td>
			<td>EX-135</td>
			<td>For step B)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro refrigerated centrifuge</td>
			<td>e.g., KUBOTA</td>
			<td align="right">3740</td>
			<td>For step 6.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator</td>
			<td>Qsonica</td>
			<td>Q125</td>
			<td>Set a microtip (#4422) on the sonicator, for step 6.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro refrigerated centrifuge</td>
			<td>e.g., TOMY</td>
			<td>MX-305</td>
			<td>For step 6.5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cooled incubator</td>
			<td>e.g., Panasonic</td>
			<td>MIR-154-PJ</td>
			<td>Set a rotary shaker inside</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotary shaker</td>
			<td>e.g., TAITEC</td>
			<td>RT-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cooled incubator&#160;</td>
			<td>e.g., TAITEC</td>
			<td>BR-43FL</td>
			<td>Set a shaker &#8220;MINI WAVE&#8221; inside, for step 7.7</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MINI WAVE</td>
			<td>AS ONE</td>
			<td>WEV-03</td>
			<td>A shaker for steps 7.7, 8.5 and 8.6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>e.g., TAITEC</td>
			<td>HB-80</td>
			<td>Set a shaker &#8220;MINI WAVE&#8221; inside, for step 8.5 and 8.6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Block incubator</td>
			<td>e.g., ASTEC</td>
			<td>BI-516S</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

With the recommended RNA template, radioactive RdRP products are observed between 20 to 30 nucleotides (nt) specifically in HeLa cells in mitotic phase after overnight exposure (Figure 1A). Typically, signal intensity of the RdRP products demonstrates two peaks that are positioned around 25 nt and 30 nt in consonance with the size distribution of the products confirmed by next generation sequencing9. In contrast to mitotic cells, HeLa cells without synchronization demonstrate almost absence of the 20&#8211;30 nt radioactive products. Oval signals, that are placed below 20 nt in all samples, are nonspecific drifts.
In case that there is only a ~30 nt product with weak signal in mitotic HeLa cells, it indicates that the RdRP reaction did not go well. For example, if MNase treatment is performed roughly and inappropriately, the signal intensity in mitotic HeLa cells decreases significantly (Figure 1B). RdRP reaction performed with old HMD solution also reduces the products (Figure 1C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57021/57021fig1.jpg" /> 
Figure 1: Representative RdRP products of endogenous TERT in mitotic HeLa cells. (A) Three representative results of the IP-RdRP assay in HeLa cells treated with nocodazole (manipulated) or DMSO (unmanipulated). The chemically synthesized 34 nt RNA was used as a template. (B) IP-RdRP assay in mitotic HeLa cells performed with inappropriate MNase treatment. (C) IP-RdRP assay in mitotic HeLa cells performed with either fresh or old HMD solution. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57021/57021fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume&#160;</td>
		</tr>
		<tr>
			<td>Micrococcal Nuclease, 20 U/&#181;L</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>Micrococcal Nuclease Buffer, 10x</td>
			<td>8 &#181;L</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>51 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: MNase reaction mix component.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume&#160;</td>
		</tr>
		<tr>
			<td>CHAPS, 0.07% (wt/vol)&#160;</td>
			<td>6 &#181;L</td>
		</tr>
		<tr>
			<td>HMD solution</td>
			<td>2 &#181;L</td>
		</tr>
		<tr>
			<td>rATP, 80 mM</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>rGTP, 8 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>rUTP, 0.4 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>rCTP, 8 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>RNase inhibitor, 40 U/&#181;L</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>1 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 2: RdRP reaction mix component.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume&#160;</td>
		</tr>
		<tr>
			<td>RNase ONE Ribonuclease, 10 U/&#181;L</td>
			<td>0.2 &#181;L</td>
		</tr>
		<tr>
			<td>Reaction Buffer, 10x</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>159.8 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 3: Ribonuclease reaction mix component.

Watch this Scientific Journal Video about Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein at JoVE.com

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

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 ...

semi-quantitative-detection-rna-dependent-rna-polymerase-activity

Nanyang Technological University

Research

JoVE Journal

Genetics

9.7K Views.  National Cancer Center Research Institute. 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.

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

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...