Dongxian Yue, Brett Robb, George Tzertzinis, Tanya Bhatia, Breton Hornblower.

RNases are commonly present on skin, hair, dust, laboratory surfaces, solutions, etc. Wear gloves and clean bench surface, pipettes etc. thoroughly before use to avoid RNase contamination. Use of nuclease- free pipette tips, tubes, glassware and reagents is strongly recommended.
1. In vitro Transcription using the T7 High-yield RNA Synthesis Kit
The DNA template for the transcription reaction is a linearized plasmid containing the Gaussia luciferase gene and a T7 promoter upstream of the coding sequence (Figure 1B).
<ol>
	<li>Thaw the components from the T7 High Yield RNA Synthesis kit and keep on ice.</li>
	<li>For uncapped RNA, set up the reaction at room temperature in the following order:</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume (μl)</td>
			<td>Final</td>
		</tr>
		<tr>
			<td>Nuclease free water*</td>
			<td>X</td>
			<td> </td>
		</tr>
		<tr>
			<td>10X Reaction Buffer</td>
			<td>2</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>ATP (100 mM)</td>
			<td>2</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>CTP (100 mM)</td>
			<td>2</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>UTP (100 mM)</td>
			<td>2</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>GTP (100 mM)</td>
			<td>2</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Template DNA *</td>
			<td>X</td>
			<td>1 μg</td>
		</tr>
		<tr>
			<td>T7 RNA Polymerase Mix</td>
			<td>2</td>
			<td> </td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20</td>
			<td> </td>
		</tr>
	</tbody>
</table>
*Add 1 μg DNA and make up the total reaction volume to 20 μl with nuclease free water. The amount of water to be added will vary based on the concentration of the template DNA.
<ol start="3">
	<li>For capped RNA: Set up the reaction at room temperature in the following order:</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume (μl)</td>
			<td>Final</td>
		</tr>
		<tr>
			<td>Nuclease free water*</td>
			<td>X</td>
			<td> </td>
		</tr>
		<tr>
			<td>10X Reaction Buffer</td>
			<td>2</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>ATP (100 mM)</td>
			<td>2</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>CTP (100 mM)</td>
			<td>2</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>UTP (100 mM)</td>
			<td>2</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>GTP (20 mM)</td>
			<td>2</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>3´-0-Me-m7G(5')ppp(5')G cap analog (ARCA)(40mM)</td>
			<td>4</td>
			<td>8mM</td>
		</tr>
		<tr>
			<td>Template DNA*</td>
			<td>X</td>
			<td>1 μg</td>
		</tr>
		<tr>
			<td>T7 RNA Polymerase Mix</td>
			<td>2</td>
			<td> </td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20</td>
			<td> </td>
		</tr>
	</tbody>
</table>
*Add 1 μg DNA and make up the total reaction volume to 20 μl with nuclease free water. The amount of water to be added will vary based on the concentration of the template DNA.
<ol start="4">
	<li>Mix well by vortexing and incubate the reactions at 37 °C for 2 hours in a dry air incubator.</li>
	<li>Using steps 1.3-1.4, synthesize capped transcripts of Cypridina luciferase (CLuc) from a linearized plasmid containing the CLuc gene. This mRNA will be co-transfected with the capped and uncapped GLuc mRNA for normalization of GLuc expression.</li>
</ol>
2. Removal of Template DNA
The transcription reactions are treated with DNase I to remove the DNA template before proceeding with purification.
<ol>
	<li>Add 70 μl nuclease free water to the transcription reactions followed by 10 μl of DNase I reaction buffer.</li>
	<li>Add 2 μl DNase I to the reactions.</li>
	<li>Incubate at 37 °C for 15 minutes in a dry air incubator.</li>
</ol>
3. Column Purification of Capped and Uncapped RNA
<ol>
	<li>Purify the capped and uncapped RNA using the MEGAclearKit as per the manufacturer's instructions (any spin column based RNA purification kit may be used).</li>
	<li>Quantify the RNA using a NanoDrop Spectrophotometer.</li>
	<li>As per the manufacturer's instructions, assess RNA sample quality using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer.</li>
</ol>
4. RNA Capping using the Vaccinia Capping System
<ol>
	<li>Take 10 μg of the purified uncapped GLuc mRNA.</li>
</ol>
A standard capping reaction can cap up to 10 μg (100nt or larger) RNA. If a larger amount of RNA needs to be capped the reaction can be scaled up accordingly.
<ol start="2">
	<li>Increase the volume to 15 μl using nuclease free water.</li>
	<li>Heat the RNA at 65 °C for 10 minutes to remove secondary structures. Place on ice for 5 minutes.</li>
	<li>To set up the capping reaction, add 2 μl 10X Capping Buffer, 1 μl GTP (10 mM), 1 μl freshly diluted S-adenosyl methionine (2 mM) and 1 μl (10 units) Vaccinia capping enzyme to the 15 μl denatured RNA.</li>
	<li>Mix well by gently vortexing and incubate the reaction at 37 °C for 30 minutes in a dry air incubator.</li>
</ol>
5. Column Purification of Capped RNA
<ol>
	<li>Purify the capped RNA using the MEGAclear Kit (any spin column based RNA purification kit may be used).</li>
	<li>As per the manufacturer's instructions, assess RNA sample quality using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer.</li>
</ol>
6. HeLa Cell Transfection using TransIT mRNA Transfection Kit
<ol>
	<li>Plate HeLa cells in a 48-well plate in 150 μl Dulbecco's Modified Eagle's Medium (DMEM), High Glucose, supplemented with 10% fetal bovine serum (0.7-2.3 × 105 cells/well).</li>
	<li>Incubate for 16-24 hours at 37 °C such that the cells are 60-90% confluent.</li>
	<li>Dilute the GLuc (capped and uncapped) and CLuc RNA to 100 ng/μl concentration.</li>
</ol>
Each transfection is done in replicates of 8.
<ol start="4">
	<li>Set up 8 eppendorf tubes. To each tube add 1 μl uncapped GLuc RNA, 1 μl capped CLuc RNA (for normalization) and 25 μl of serum free DMEM. Mix well.</li>
	<li>Set up 8 eppendorf tubes. To each tube add 1 μl capped GLuc RNA (cap analog), 1 μl capped CLuc RNA (for normalization) and 25 μl of serum free DMEM. Mix well.</li>
	<li>Set up 8 eppendorf tubes. To each tube add 1 μl capped GLuc RNA (Vaccinia), 1 μl capped CLuc RNA (for normalization) and 25 μl of serum free DMEM. Mix well.</li>
	<li>Add 0.2 μl mRNA Boost Reagent to the first set of 8 tubes and mix.</li>
	<li>Add 0.2 μl TransIT mRNA Reagent to the first set of 8 tubes and mix.</li>
	<li>Incubate at room temperature for 2-5 minutes. (Do not incubate longer than 5 minutes).</li>
	<li>Add the transfection mixture drop wise to the wells containing HeLa cell cultures (refer Figure 3 for plate set-up).</li>
	<li>Repeat steps 7 through 10 with the 2nd and 3rd sets of 8 tubes.</li>
	<li>Incubate overnight (16 hours) at 37 °C in 5% CO2.</li>
</ol>
7. Gaussia Luciferase (GLuc) Assay
Assay the supernatant from each well for GLuc activity using the BioLux Gaussia Luciferase Assay Kit as follows:
<ol>
	<li>Prepare fresh assay solution by adding 15 μl of BioLux GLuc Substrate to 1.5 ml of BioLux GLuc Assay Buffer (50 μl assay solution required per sample). Prepare enough for samples and for priming luminometer injector (refer to manufacturer's instructions).</li>
	<li>Mix well by inverting the tube several times (do not vortex).</li>
</ol>
Luminescence is measured using the Centro LB 960 microplate luminometer from Berthold Technologies.
<ol start="3">
	<li>Set the luminometer to 50 μl injection volume and 2-10 seconds integration.</li>
	<li>Add 10 μl of the cell culture supernatant from each well into a 96-well black plate.</li>
	<li>Prime the injector with the assay solution and proceed with measurement of luminescence.</li>
	<li>Values obtained from untransfected wells will be used as negative controls.</li>
</ol>
8. Cypridina Luciferase (CLuc) Assay
Assay the supernatant from each well for CLuc activity using the BioLux Cypridina Luciferase Assay Kit as follows:
<ol>
	<li>Prepare fresh reconstituted substrate (100x) according to instructions given in the kit manual.</li>
	<li>Thaw the BioLux Cypridina Luciferase Assay Buffer and mix well (protect from light).</li>
	<li>To prepare the CLuc assay solution, add 15 μl of the reconstituted substrate (100x) to 1.5 ml of BioLux Cypridina Luciferase Assay Buffer (50 μl assay solution required per sample). Prepare enough for samples and for priming luminometer injector (refer to manufacturer's instructions).</li>
	<li>Mix well by inverting the tube several times (do not vortex).</li>
	<li>Keep the solution at room temperature for 30 minutes (protect from light).</li>
</ol>
Luminescence is measured using the Centro LB 960 microplate luminometer from Berthold Technologies.
<ol start="6">
	<li>Set the luminometer to 50 μl injection, 1-2 seconds delay and 2-10 seconds integration.</li>
	<li>Add 10 μl of the cell culture supernatant from each well into a 96-well black plate.</li>
	<li>Prime the injector with the CLuc assay solution and measure luminescence.</li>
	<li>Values obtained from untransfected wells will be used as negative controls.</li>
</ol>
9. Representative Results
The T7 High Yield RNA Synthesis Kit can produce up to 180 μg uncapped RNA and 40 to 50 μg of capped RNA per 20 μl reaction. When analyzed on the Agilent 2100 Bioanalyzer, good quality, intact RNA should show a single, sharp peak representing the RNA transcript. A broad peak or multiple peaks indicate RNA degradation. It is also important to verify the size of the RNA. RNA transcripts of a longer length than expected may be due to incomplete digestion of the template plasmid DNA. RNA degradation and lower yield are usually a result of contaminants introduced into the reaction from the template DNA. Figure 2 is an example of the electropherogram and gel image obtained after running high quality, intact RNA on the Bioanalyzer.
Transfection of HeLa cells with both capped and uncapped GLuc RNA is followed by incubation and assaying cell culture supernatants for luciferase activity. The 5' cap structure is important for protecting the RNA against exonuclease degradation8 and for promoting translation initiation9 of the mRNA. Therefore, RNA capping is an essential step for transfection experiments. The co-transcriptional (cap analog) method yields approximately 40 μg RNA that is 80% capped. The protocol utilizing Vaccinia capping enzyme caps up to 10 μg RNA (100 nt or larger) per reaction with nearly 100% efficiency.
Figure 4 depicts the difference in luciferase expression between cultures transfected with capped and uncapped RNA samples. It can be clearly seen that cell cultures transfected with capped RNA show much higher expression of luciferase as compared to those transfected with uncapped RNA (which show no luciferase activity). In addition, Figure 4 also validates that both capping techniques, using cap analog and using the Vaccinia capping enzyme, successfully produce functional, capped RNA transcripts that can be translated into protein. The two tailed p-value obtained from the t-test was 0.2583 which indicates that there is no significant statistical difference between the luminescence data from the two capping methods.
<img alt="T7 transcription process diagram; DNA to RNA sequence conversion with T7 promoter and runoff transcript." src="/files/ftp_upload/3702/3702fig1A.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1.">
Figure 1A. RNA Synthesis by T7 RNA Polymerase
 
<img alt="Plasmid map diagram of pCMV-GLuc, 5764 bp, shows restriction sites and functional gene regions." src="/files/ftp_upload/3702/3702fig1B.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2."> 
Figure 1B. pCMV-GLuc vector containing GLuc gene and T7 promoter.
 
<img alt="RNA cap analysis; electropherograms and gel electrophoresis; uncapped/capped RNA expression." src="/files/ftp_upload/3702/3702fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2."> 
Figure 2. Bioanalyzer electropherograms and gel- image of capped and uncapped RNA . The X-axis is the size of the RNA in nucleotides (nt) and the Y-axis represents fluorescence units (FU). A) Electropherogram of GLuc mRNA (uncapped) after purification. B) Electropherogram of GLuc mRNA (capped using Vaccinia capping enzyme). C) Electropherogram of GLuc mRNA (capped co-transcriptionally using cap analog). D) Gel-like image generated by the Bioanalyzer. The 'L' lane is the RNA ladder, Lane 1 is uncapped GLuc mRNA, Lane 2 is GLuc mRNA capped using the cap analog and Lane 3 is the GLuc RNA capped using Vaccinia capping enzyme.
 
<img alt="Grid diagram of cell samples in gene expression experiment; Gluc, CLuc, capped, uncapped." src="/files/ftp_upload/3702/3702fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3."> 
Figure 3. Example of plate set-up for HeLa cell transfection . Each well consists of plated HeLa cells that are 60-90% confluent. The transfection mix consisting of the appropriate GLuc RNA (uncapped, capped with cap analog or capped with Vaccinia capping enzyme), capped CLuc RNA (for normalization), mRNA Boost Reagent and TransIT mRNA Reagent is added to each well. Each transfection is done in replicates of 8 (same colored wells in the figure).
 
 
<img alt="Bar chart comparing normalized luminescence for GLuc RNA caps, showcasing experimental results." src="/files/ftp_upload/3702/3702fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4."> 
Figure 4.GLuc expression in HeLa cells. Purified capped and uncapped GLuc mRNA was transfected into HeLa cells and incubated overnight (16 hrs) at 37 °C. Cell culture supernatants from each well were assayed for GLuc and CLuc activity and luminescence values were recorded. The GLuc luminescence values were normalized to the luminescence values of capped CLuc RNA.
<img alt="Normalized GLuc luminescence formula; equation illustrating luminescence value ratio calculation." src="/files/ftp_upload/3702/3702eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1">
* Luminescence from untransfected cells was subtracted from GLuc and CLuc values before normalization.
 
Trademarks
BioLux is a trademark of New England Biolabs, Inc.
TransIT is a registered trademark of Mirus Bio LLC.
MEGAclear is a trademark of Ambion.

<ol>
	<li>Lazarev, D., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+splicing+and+transcription+are+not+sufficient+to+enhance+splicing+efficiency.">Concurrent splicing and transcription are not sufficient to enhance splicing efficiency.</a> RNA. 13, 1546-1557 (2007).</li><li>Donze, O., Picard, D. <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+mammalian+cells+using+siRNAs+synthesized+with+T7.">RNA interference in mammalian cells using siRNAs synthesized with T7.</a> RNA Polymerase. Nucl. Acids Res. 30, 10-46 (2002).</li><li>Van Gelder, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Nati. Acad. Sci. U.S.A. 87, 1663-1667 (1990).</li><li>Schneider, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+T7+RNA+polymerase+based+in+vitro+linear+RNA+amplification+for+use+in+microarray+experiments.">Systematic analysis of T7 RNA polymerase based in vitro linear RNA amplification for use in microarray experiments.</a> BMC Genomics. 5, 29-29 (2004).</li><li>Conry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+messenger+RNA+polynucleotide+vaccine+vector.">Characterization of a messenger RNA polynucleotide vaccine vector.</a> Cancer Research. 55, 1397-1400 (1995).</li><li>Huang, F., Wang, G., Coleman, T., Li, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+adenosine+derivatives+as+transcription+initiators+and+preparation+of+5â&#x20AC;²+fluorescein-+and+biotin-labeled+RNA+through+one-step+transcription.">Synthesis of adenosine derivatives as transcription initiators and preparation of 5â&#x20AC;² fluorescein- and biotin-labeled RNA through one-step transcription.</a> RNA. 9, 1562-1570 (2003).</li><li>Warren, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+reprogramming+to+pluripotency+and+directed+differentiation+of+human+cells+with+synthetic+modified+mRNA.">Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.</a> Cell Stem Cell. 7, 618-630 (2010).</li><li>Furuichi, Y., LaFiandra, A., Shatkin, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5'-Terminal+structure+and+mRNA+stability.">5'-Terminal structure and mRNA stability.</a> Nature. 266, 235-239 (1977).</li><li>Topisirovic, I., Svitkin, Y. V., Sonenberg, N., Shatkin, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap+and+cap+binding+proteins+in+the+control+of+gene+expression.">Cap and cap binding proteins in the control of gene expression.</a> Wiley Interdisciplinary Reviews. 2, 277-298 (2011).</li><li>Lewis, J. D., Izaurflde, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Cap+Structure+in+RNA+Processing+and+Nuclear+Export.">The Role of the Cap Structure in RNA Processing and Nuclear Export.</a> Eur. J. Biochem. 247, 461-469 (1997).</li><li>Inoue, K., Ohno, M., Sakamoto, H., Shimura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+cap+structure+on+pre-mRNA+splicing+in+Xenopus+oocyte+nuclei.">Effect of the cap structure on pre-mRNA splicing in Xenopus oocyte nuclei.</a> Genes Dev. 3, 1472-1479 (1989).</li><li>Martin, S. A., Paoletti, E., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+mRNA+guanylyltransferase+and+mRNA+(guanine+7-)methyltransferase+from+vaccinia+virus.">Purification of mRNA guanylyltransferase and mRNA (guanine 7-)methyltransferase from vaccinia virus.</a> J. Biol. Chem. 250, 9322-9329 (1975).</li><li>Martin, S. A., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+RNA+by+mRNA+guanylytransferase+and+mRNA+(guanine-7-)methyl-transferase+from+vaccinia+virions.">Modification of RNA by mRNA guanylytransferase and mRNA (guanine-7-)methyl-transferase from vaccinia virions.</a> J. Biol. Chem. 250, 9330-9335 (1975).</li><li>Shuman, S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+use+of+vaccinia+virus+mRNA+capping+enzyme.">Purification and use of vaccinia virus mRNA capping enzyme.</a> Meth. Enzymol. 181, 170-180 (1990).</li><li>Thiel, V., Herold, J., Scelle, B., Siddell, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+RNA+transcribed+in+vitro+from+a+cDNA+copy+of+the+human+coronavirus+genome+cloned+in+vaccinia+virus.">Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus.</a> J. Gen. Virol. 82, 1273-1281 (2001).</li><li>Marcotrigiano, J., Gingras, A. -. C., Sonenberg, N., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocrystal+Structure+of+the+Messenger+RNA+5â&#x20AC;²+Cap-Binding+Protein+(eIF4E)+Bound+to+7-methyl-GDP.">Cocrystal Structure of the Messenger RNA 5â&#x20AC;² Cap-Binding Protein (eIF4E) Bound to 7-methyl-GDP.</a> Cell. 89, 951-961 (1997).</li><li>Stepinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+properties+of+mRNAs+containing+the+novel+"anti-reverse"+cap+analogs+7-methyl+(3'-O-methyl)GpppG+and+7-methyl+(3'-deoxy)+GpppG.">Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl (3'-O-methyl)GpppG and 7-methyl (3'-deoxy) GpppG.</a> RNA. 7, 1486-1495 (2001).</li><li>Kuge, H., Brownlee, G. G., Gershon, P. D., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap+ribose+methylation+of+c-mos+mRNA+stimulates+translation+and+oocyte+maturation+in+Xenopus+laevis.">Cap ribose methylation of c-mos mRNA stimulates translation and oocyte maturation in Xenopus laevis.</a> Nucl. Acids Res. 26, 3208-3214 (1998).</li><li>Schagat, T., Paguio, A., Kopish, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalizing+Genetic+Reporter+Assays:+Approaches+and+Considerations+for+Increasing+Consistency+and+Statistical+Significance.">Normalizing Genetic Reporter Assays: Approaches and Considerations for Increasing Consistency and Statistical Significance.</a> Cell Notes. 17, 9-12 (2007).</li><li>Barbosa, E., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+(nucleoside-2'-)+-+methyltransferase+from+Vaccinia+Virus.">mRNA (nucleoside-2'-) - methyltransferase from Vaccinia Virus.</a> J. Biol. Chem. 253, 7698-7702 (1978).</li><li>Lockless, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+capped+RNA+substrates+by+VP39,+the+vaccinia+virus-encoded+mRNA+cap-specific+2'-O-methyltransferase.">Recognition of capped RNA substrates by VP39, the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase.</a> Biochemistry. 37, 8564-8574 (1998).</li></ol>

All authors are employees of New England Biolabs, Inc.

In vitro transcription is a useful method for obtaining high yields of RNA for a variety of applications. The major advantage of using the T7 High Yield RNA Synthesis Kit is that its formulation has been optimized to achieve high yields of RNA. In addition, the reagents provided in the kit are free of contaminating nucleases, resulting in synthesis of high quality, intact RNA transcripts. The kit is designed for high stability and flexibility such that it can be used for synthesizing mRNA, dye labeled RNA, high specific activity radiolabeled probes and capped RNA. The kit manual includes a list of specific protocols and additional materials required for each of these applications. It also includes technical information on using PCR products and synthetic DNA oligonucleotides as templates for transcription. It is important to note that the transcription kit can only utilize DNA templates containing a T7 promoter.
The method described in this article demonstrates post-transcriptional RNA capping using Vaccinia capping enzyme, as well as co-transcriptional capping using RNA cap structure analog. We have shown that both methods synthesize capped RNA that is functionally active post-transfection. The 5' cap structure improves stability of the RNA and translation efficiency, and hence is important for microinjection11 and transfection7 experiments. The co-transcriptional capping method generates 40-50 &mu;g of ~80% capped RNA. It is important to note that using 3'-0-Me-m7G(5')ppp(5')G RNA cap analog (ARCA), which is blocked at the 3'-hydroxyl of the m7G ensures incorporation of the cap in the correct orientation17. To cap larger quantities of RNA it is possible to scale up the standard reactions for both capping methods. However, using the Vaccinia Capping System would be a more feasible option cost-wise since co-transcriptional capping utilizes cap analog, which is a relatively expensive component. The Vaccinia Capping System is also a better method since it has capping efficiency of almost 100%.
The RNA capping methods described above synthesize RNA with a cap 0 structure at the 5' end. Cap 1 structure involves additional methylation at the 2'-O position of the ribose sugar of the first nucleotide at the ' end of the RNA. The cap 1 structure has been reported to enhance RNA translation efficiency18 and can be incorporated by using 2'-O- Methyltransferase20, 21.
The method described in this paper can be used to transcribe, cap and transfect any desired mRNA. The luciferases GLuc and CLuc were specifically used in this protocol due to their many advantages. They are directly secreted into the cell medium, therefore avoiding need for cell lysis. Also, they generate high bioluminescent signal intensity, and the activity assays are highly sensitive and easy to perform. It is important to note that the GLuc expression in the cells can be influenced by various factors other than the functionality of the RNA itself. Some examples are pipetting errors, varying cell numbers per well, transfection efficiency etc. Data normalization is used to account for these factors. In our protocol, we co-transfect CLuc RNA as an internal control with the GLuc RNA. When GLuc luminescence is divided by the CLuc control values, it gets normalized for the errors and variability in transfection19.

<table><tbody><tr><td>T7 High Yield RNA Synthesis Kit</td><td>New England Biolabs</td><td>E2040S</td><td></td></tr><tr><td>3&amp;rsquo;-0-Me-m&lt;sup&gt;7&lt;/sup&gt;G(5&amp;rsquo;)ppp(5&amp;rsquo;)G RNA Cap Structure Analog </td><td>New England Biolabs</td><td>S1411S</td><td></td></tr><tr><td>DNase I (RNase-free)</td><td>New England Biolabs</td><td>M0303S</td><td></td></tr><tr><td>Vaccinia Capping System</td><td>New England Biolabs</td><td>M2080S</td><td></td></tr><tr><td>MEGAclear Kit</td><td>Ambion</td><td>AM1908</td><td></td></tr><tr><td>NanoDrop Spectrophotometer</td><td>Thermo Fisher Scientific, Inc.</td><td></td><td></td></tr><tr><td>Agilent RNA 6000 Nano Kit</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2939AA</td><td></td></tr><tr><td>&lt;em&gt;Trans&lt;/em&gt;IT mRNA Transfection Kit </td><td>Mirus Bio LLC</td><td>MIR2250</td><td></td></tr><tr><td>HyClone* Classical Liquid Media: Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium, High Glucose</td><td>Thermo Fisher Scientific, Inc.</td><td>SH30285.01</td><td></td></tr><tr><td>HyClone Non-essential Amino Acids</td><td>Thermo Fisher Scientific, Inc.</td><td>SH30238.01</td><td></td></tr><tr><td>Centro LB 960 </td><td>Berthold Technologies</td><td></td><td></td></tr><tr><td>BioLux&lt;em&gt; Gaussia&lt;/em&gt; Luciferase Assay Kit</td><td>New England Biolabs</td><td>E3300S</td><td></td></tr><tr><td>BioLux &lt;em&gt;Cypridina&lt;/em&gt; Luciferase Assay Kit</td><td>New England Biolabs</td><td>E3309S</td><td></td></tr><tr><td>RNase Inhibitor, Murine </td><td>New England Biolabs</td><td>M0314</td><td></td></tr><tr><td>RNase Inhibitor, Human Placenta</td><td>New England Biolabs</td><td>M0307</td><td></td></tr><tr><td>pCMV-GLuc Control Plasmid</td><td>New England Biolabs</td><td>N8081</td><td></td></tr></tbody></table>

in vitro transcription and capping of gaussia luciferase mrna followed by hela cell transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

Watch this Scientific Journal Video about In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection at JoVE.com

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

in-vitro-transcription-capping-gaussia-luciferase-mrna-followed-hela

Research

JoVE Journal

Biology

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

18.2K 次观看.  New England Biolabs. This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

视频: In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

科研

遗传学

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

一种基于报告基因的细胞检测，用于监测剪接效率

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

生物化学

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

hiv 转录和拼接中延迟逆转剂的高通量体外评价

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

免疫与感染

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

生物学

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...