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 (&mu;l)</td>
			<td>Final</td>
		</tr>
		<tr>
			<td>Nuclease free water*</td>
			<td>X</td>
			<td>&nbsp;</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 &mu;g</td>
		</tr>
		<tr>
			<td>T7 RNA Polymerase Mix</td>
			<td>2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
*Add 1 &mu;g DNA and make up the total reaction volume to 20 &mu;l 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 (&mu;l)</td>
			<td>Final</td>
		</tr>
		<tr>
			<td>Nuclease free water*</td>
			<td>X</td>
			<td>&nbsp;</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&acute;-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 &mu;g</td>
		</tr>
		<tr>
			<td>T7 RNA Polymerase Mix</td>
			<td>2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
*Add 1 &mu;g DNA and make up the total reaction volume to 20 &mu;l 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 &deg;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 &mu;l nuclease free water to the transcription reactions followed by 10 &mu;l of DNase I reaction buffer.</li>
	<li>Add 2 &mu;l DNase I to the reactions.</li>
	<li>Incubate at 37 &deg;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 &mu;g of the purified uncapped GLuc mRNA.</li>
</ol>
A standard capping reaction can cap up to 10 &mu;g (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 &mu;l using nuclease free water.</li>
	<li>Heat the RNA at 65 &deg;C for 10 minutes to remove secondary structures. Place on ice for 5 minutes.</li>
	<li>To set up the capping reaction, add 2 &mu;l 10X Capping Buffer, 1 &mu;l GTP (10 mM), 1 &mu;l freshly diluted S-adenosyl methionine (2 mM) and 1 &mu;l (10 units) Vaccinia capping enzyme to the 15 &mu;l denatured RNA.</li>
	<li>Mix well by gently vortexing and incubate the reaction at 37 &deg;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 &mu;l Dulbecco's Modified Eagle's Medium (DMEM), High Glucose, supplemented with 10% fetal bovine serum (0.7-2.3 &times; 105 cells/well).</li>
	<li>Incubate for 16-24 hours at 37 &deg;C such that the cells are 60-90% confluent.</li>
	<li>Dilute the GLuc (capped and uncapped) and CLuc RNA to 100 ng/&mu;l 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 &mu;l uncapped GLuc RNA, 1 &mu;l capped CLuc RNA (for normalization) and 25 &mu;l of serum free DMEM. Mix well.</li>
	<li>Set up 8 eppendorf tubes. To each tube add 1 &mu;l capped GLuc RNA (cap analog), 1 &mu;l capped CLuc RNA (for normalization) and 25 &mu;l of serum free DMEM. Mix well.</li>
	<li>Set up 8 eppendorf tubes. To each tube add 1 &mu;l capped GLuc RNA (Vaccinia), 1 &mu;l capped CLuc RNA (for normalization) and 25 &mu;l of serum free DMEM. Mix well.</li>
	<li>Add 0.2 &mu;l mRNA Boost Reagent to the first set of 8 tubes and mix.</li>
	<li>Add 0.2 &mu;l 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 &deg;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 &mu;l of BioLux GLuc Substrate to 1.5 ml of BioLux GLuc Assay Buffer (50 &mu;l 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 &mu;l injection volume and 2-10 seconds integration.</li>
	<li>Add 10 &mu;l 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 &mu;l of the reconstituted substrate (100x) to 1.5 ml of BioLux Cypridina Luciferase Assay Buffer (50 &mu;l 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 &mu;l injection, 1-2 seconds delay and 2-10 seconds integration.</li>
	<li>Add 10 &mu;l 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 &mu;g uncapped RNA and 40 to 50 &mu;g of capped RNA per 20 &mu;l 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 &mu;g RNA that is 80% capped. The protocol utilizing Vaccinia capping enzyme caps up to 10 &mu;g 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="Figure 1." src="/files/ftp_upload/3702/3702fig1A.jpg" />
Figure 1A. RNA Synthesis by T7 RNA Polymerase
 
<img alt="Figure 2." src="/files/ftp_upload/3702/3702fig1B.jpg" /> 
Figure 1B. pCMV-GLuc vector containing GLuc gene and T7 promoter.
 
<img alt="Figure 2." src="/files/ftp_upload/3702/3702fig2.jpg" /> 
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="Figure 3." src="/files/ftp_upload/3702/3702fig3.jpg" /> 
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="Figure 4." src="/files/ftp_upload/3702/3702fig4.jpg" /> 
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 &deg;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="Equation 1" src="/files/ftp_upload/3702/3702eq1.jpg" />
* 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.

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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 border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>T7 High Yield RNA Synthesis Kit</td>
 <td>New England Biolabs</td>
 <td>E2040S</td>
 <td></td>
 </tr>
 <tr>
 <td>3&acute;-0-Me-m7G(5')ppp(5')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 Scientific</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>TransIT mRNA Transfection Kit </td>
 <td>Mirus Bio LLC</td>
 <td>MIR2250</td>
 <td></td>
 </tr>
 <tr>
 <td>HyClone* Classical Liquid Media: Dulbecco's Modified Eagle's Medium, High Glucose</td>
 <td>Thermo Scientific</td>
 <td>SH30285.01</td>
 <td></td>
 </tr>
 <tr>
 <td>HyClone Non-essential Amino Acids</td>
 <td>Thermo Scientific</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 Gaussia Luciferase Assay Kit</td>
 <td>New England Biolabs</td>
 <td>E3300S</td>
 <td></td>
 </tr>
 <tr>
 <td>BioLux Cypridina 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.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

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

18.1K Aufrufe.  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.

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikörper Profiling von Luciferase Immunpräzipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Forschung

Biologie

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Single Cell Transfektion in Hühnerembryonen

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

Die Messung der Kinetik der mRNA Transkription in einzelnen lebenden Zellen

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

In vivo Leber Endozytose durch Reinigung von Leberzellen durch Leberperfusion Gefolgt

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Analyse von Einzel-Zell-Gene Transkription durch RNA-Fluorescent<em&gt; In Situ</em&gt; Hybridisierung (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

Die Analyse der RNA-Prozessierung Reaktionen mit Handy Free Systems: 3 &#39;Ende Spaltung von prä-mRNA-Substrate<em&gt; In-vitro-</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

In vitro Synthese von modifizierten mRNA zur Induktion der Proteinexpression in menschlichen Zellen

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Bewertung der Selective mRNA Translation in Säugerzellen durch Polysomen Profilieren

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Überwachung Endoplasmatischen Reticulum Calciumhomöostase Mit ein<em&gt; Gaussia</em&gt; Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...