Name: a nonsequencing approach for the rapid detection of rna editing
Uploaded: 2022-04-21T13:20:00
Description: Watch this Scientific Journal Video about A Nonsequencing Approach for the Rapid Detection of RNA Editing at JoVE.com

This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17H02204 and 18K19288). Ruchika was financially supported by the Japanese government (MEXT scholarship). We thank Ms. Radhika Biyani (Takagi Laboratory, JAIST) and Dr. Kirti Sharma (BioSeeds Corporation) for help with electrophoresis-related experiments.

1. Optimization of the target fragment
NOTE: Four edited genes were used in the development of this protocol, including two nuclear genes (AT2G16586 and AT5G02670) from A. thaliana, and the genes encoding blue fluorescent protein (BFP) and enhanced green fluorescent protein (EGFP) expressed in HEK293 cells.
<ol>
	<li>To identify gene fragments with different melting profiles, representing edited versus nonedited regions, generate predicted melting curves using the u...

<ol>
	<li>Chateigner-Boutin, A. L., Small, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+high-throughput+method+for+the+detection+and+quantification+of+RNA+editing+based+on+high-resolution+melting+of+amplicons.">A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons.</a> Nucleic Acids Research. 35 (17), 114 (2007).</li><li>Chen, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+PCR+method+for+the+quantitative+analysis+of+RNA+editing+at+specific+sites.">A real-time PCR method for the quantitative analysis of RNA editing at specific sites.</a> Analytical Biochemistry. 375 (1), 46-52 (2008).</li><li>Barbon, A., Vallini, I., La Via, L., Marchina, E., Barlati, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+RNA+editing:+a+molecular+analysis+of+GluR2,+GluR5+and+GluR6+in+human+brain+tissues+and+in+NT2+cells+following+in+vitro+neural+differentiation.">Glutamate receptor RNA editing: a molecular analysis of GluR2, GluR5 and GluR6 in human brain tissues and in NT2 cells following in vitro neural differentiation.</a> Molecular Brain Research. 117 (2), 168-178 (2003).</li><li>Gallo, A., Thomson, E., Brindle, J., O'Connell, M. A., Keegan, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-processing+events+in+mRNAs+identified+by+DHPLC+analysis.">Micro-processing events in mRNAs identified by DHPLC analysis.</a> Nucleic Acids Research. 30 (18), 3945-3953 (2002).</li><li>Schiffer, H. H., Heinemann, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Quantitative+method+to+detect+RNA+editing+events.">A Quantitative method to detect RNA editing events.</a> Analytical Biochemistry. 276 (2), 257-260 (1999).</li><li>Burns, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+serotonin-2C+receptor+G-protein+coupling+by+RNA+editing.">Regulation of serotonin-2C receptor G-protein coupling by RNA editing.</a> Nature. 387 (6630), 303-308 (1997).</li><li>Marian, 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=The+bottleneck+in+genetic+testing.">The bottleneck in genetic testing.</a> Circulation Research. 117 (7), 586-588 (2015).</li><li>Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+biology:+Not+drowning+but+waving.">Genome biology: Not drowning but waving.</a> Cell. 154 (5), 951-952 (2013).</li><li>Jones, B. M., Knapp, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+temperature+gradient+electrophoresis+for+detection+of+single+nucleotide+polymorphisms.">Temporal temperature gradient electrophoresis for detection of single nucleotide polymorphisms.</a> Methods in Molecular Biology. 578, 153-165 (2009).</li><li>Riesner, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-gradient+gel+electrophoresis+of+nucleic+acids:+analysis+of+conformational+transitions,+sequence+variations,+and+protein-nucleic+acid+interactions.">Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions.</a> Electrophoresis. 10 (5-6), 377-389 (1989).</li><li>Biyani, M., Nishigaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hundred-fold+productivity+of+genome+analysis+by+introduction+of+micro-temperature+gradient+gel+electrophoresis.">Hundred-fold productivity of genome analysis by introduction of micro-temperature gradient gel electrophoresis.</a> Electrophoresis. 22 (1), 23-28 (2001).</li><li>Rathore, H., Biyani, R., Kato, H., Takamura, Y., Biyani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palm-size+and+one-inch+gel+electrophoretic+device+for+reliable+and+field-applicable+analysis+of+recombinase+polymerase+amplification.">Palm-size and one-inch gel electrophoretic device for reliable and field-applicable analysis of recombinase polymerase amplification.</a> Analytical Methods. 11 (39), 4969-4976 (2019).</li><li>Dwight, Z., Palais, R., Wittwer, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=uMELT:+prediction+of+high-resolution+melting+curves+and+dynamic+melting+profiles+of+PCR+products+in+a+rich+web+application.">uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application.</a> Bioinformatics. 27 (7), 1019-1020 (2011).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. <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+RNA+using+TRIzol+(TRI+Reagent).">Purification of RNA using TRIzol (TRI Reagent).</a> Cold Spring Harbor Protocols. 2010 (6), (2010).</li><li>Bhakta, S., Sakari, M., Tsukahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+editing+of+BFP,+a+point+mutant+of+GFP,+using+artificial+APOBEC1+deaminase+to+restore+the+genetic+code.">RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code.</a> Scientific Reports. 10 (1), 17304 (2020).</li><li>Azad, T. A., Qulsum, U., Tsukahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+activity+of+adenosine+deaminase+acting+on+RNA+(ADARs)+isoforms+for+correction+of+genetic+code+in+gene+therapy.">Comparative activity of adenosine deaminase acting on RNA (ADARs) isoforms for correction of genetic code in gene therapy.</a> Current Gene Therapy. 19 (1), 31-39 (2019).</li><li>Ruchika, O. C., Sakari, M., Tsukahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+U-To-C+RNA+editing+events+for+nuclear+genes+in+Arabidopsis+thaliana.">Genome-wide identification of U-To-C RNA editing events for nuclear genes in Arabidopsis thaliana.</a> Cells. 10 (3), 635 (2021).</li><li>Tsukahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+U-to-C+RNA+editing+affects+the+mRNA+stability+of+nuclear+genes+in+Arabidopsis+thaliana.">The U-to-C RNA editing affects the mRNA stability of nuclear genes in Arabidopsis thaliana.</a> Biochemical and Biophysical Research Communications. 571, 110-117 (2021).</li></ol>

RNA editing plays an important role in biology; however, current methods of detecting RNA editing, such as chromatography and sequencing, present several challenges due to their high cost, excessive time requirements, and complexity. The protocol described here is a simple, rapid, and cost-effective method of detecting RNA editing that uses a portable, microsized, TGGE-based system. This system can be used to differentiate between edited and nonedited genes prior to Sanger sequencing. Specifically, edited and nonedited g...

Single nucleotide variants (SNVs) in genomic RNA, including A-to-I, C-to-U, and U-to-C variants, can indicate RNA editing events. However, the detection of SNVs in RNA remains a technically challenging task. Conventionally, the ratio of edited to nonedited RNA is determined by direct sequencing, allele-specific real-time polymerase chain reaction (PCR), or denaturing high-performance liquid chromatography (HPLC) approaches1,2,3,4,5,6. However, these approaches are not particularly time- or cost-effective, and their low accuracies, caused by high levels of noise, pose technological bottlenecks for RNA-based SNV detection7,8. Here, we describe a protocol based on temperature gradient gel electrophoresis (TGGE) to identify single nucleotide polymorphisms (SNPs) as an alternative method that eliminates the need for direct RNA sequencing approaches to RNA editing analyses.
Electrophoresis is a preferred method for the separation and analysis of biomolecules in life science laboratories. TGGE enables the separation of double-stranded DNA fragments that are the same size but have different sequences. The technique relies on sequence-related differences in the melting temperatures of DNA fragments and subsequent changes in their mobilities in porous gels with a linear temperature gradient9,10. Melting of DNA fragments generates specific melting profiles. Once the domain with the lowest melting temperature reaches the corresponding temperature at a particular position in the gel, the transition from a helical structure to a partially melted structure occurs, and migration of the molecule will practically halt. Therefore, TGGE utilizes both mobility (size information) and temperature-induced structural transitions of DNA fragments (sequence-dependent information), making it a powerful approach to the characterization of DNA fragments. The feature points in a TGGE melting pattern, which correspond to three structural transitions of the DNA molecule, are the strand initial-dissociation point, the strand mid-dissociation point, and the strand end-dissociation point (Figure 1). Sequence variations within domains, even single-base differences, affect the melting temperature, hence, molecules with different sequences will show discrete melting (denaturation) patterns in TGGE analyses. Therefore, TGGE can be used to analyze SNVs in genomic RNA and can be an invaluable high-throughput method of detecting RNA editing. This high-throughput gain is lost when traditional gel electrophoresis-based TGGE is used. However, a miniaturized version of TGGE, named microTGGE (&#181;TGGE), can be used to shorten the gel electrophoretic time and accelerate the analysis with a 100-fold increase in productivity11. The simplicity and compactness of the &#181;TGGE method have been improved by the introduction of PalmPAGE12, a field-applicable, handheld, and affordable gel electrophoresis system.
Here, a new TGGE-based protocol is used to examine three types of RNA editing sites (A-to-I, C-to-U, and U-to-C) in four genes, including two from Arabidopsis thaliana tissues and two expressed in mammalian HEK293 cells (Figure 1A). The protocol integrates the use of PalmPAGE (hardware), a portable system for the rapid detection of RNA editing, and uMelt (software)13. With an average run-time of 15-30 min, this protocol enables rapid, reliable, and easy identification of RNA editing without the need for direct RNA sequencing approaches.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2U ExoI</td>
			<td>Takara</td>
			<td>2650A</td>
			<td>Exonuclease I</td>
		</tr>
		<tr>
			<td>40(w/v)%-acrylamide/bis (19:1)</td>
			<td>Thermo fisher</td>
			<td>AM9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Thermo Fisher</td>
			<td>17874</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Mini spin</td>
			<td>eppendorf</td>
			<td>5452000034</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital dry bath/ block heater</td>
			<td>Thermo fisher</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold Taq Polymerase Master mixture</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>LATaq DNA polymerase</td>
			<td>TAKARA</td>
			<td>RR002A</td>
			<td>Taq Polymerase</td>
		</tr>
		<tr>
			<td>micro-TGGE&#160; cassette holder</td>
			<td>BioSeeds Corp.</td>
			<td>BS-GE-CH</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-TGGE apparatus</td>
			<td>Lifetech Corp.</td>
			<td>TG</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-TGGE gel cassette</td>
			<td>BioSeeds Corp.</td>
			<td>BS-TGGE-C</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000</td>
			<td>Thermo fisher</td>
			<td>ND-1000</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>Plant Rneasy Mini kit</td>
			<td>Qiagen</td>
			<td>74904</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace Master Mix</td>
			<td>TOYOBO</td>
			<td>TRT101</td>
			<td>M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase</td>
		</tr>
		<tr>
			<td>Rneasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Shrimp Alkaline Phosphatase</td>
			<td>Takara</td>
			<td>2660B</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Gold nucleic acid gel stain</td>
			<td>Thermo fisher</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE buffer</td>
			<td>Thermo fisher</td>
			<td>B52</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Nacalai tesque</td>
			<td>33401-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea, Nuclease and protease tested</td>
			<td>Nacalai tesque</td>
			<td>35940-65</td>
			<td></td>
		</tr>
	</tbody>
</table>

a nonsequencing approach for the rapid detection of rna editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Use of &#181;TGGE to identify single nucleotide base changes in RNA editing events 
Four edited genes were used for this protocol (Table 1), including the BFP gene produced in HEK293 cells (with C-to-U RNA editing by the deaminase enzymes of apolipoprotein B mRNA editing enzyme complex; APOBEC115), the EGFP gene containing the ochre stop codon (TAA) produced in HEK293 cells (with A-to-I RNA editing by adenosine deaminase acting on RNA 1; ADAR1<sup class="xref...

Watch this Scientific Journal Video about A Nonsequencing Approach for the Rapid Detection of RNA Editing at JoVE.com

A Nonsequencing Approach for the Rapid Detection of RNA Editing

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...

RNA editing play a critical role in human disease. Scientist are finding endless application in the medicines. This protocol demonstrate the application of portable temperature electrophoresis technology as a non sequencing approach for the rapid and reliable identification of RNA modification in RNA editing without the need of direct RNA sequencing.The micro tTG electrophoresis based approach was used to characterize the differences between the melting profile of four edited RNA fragments and their corresponding non-edited fragments. The pattern similarity scores were used to assess the reproducibility of the method. This platform enable the detection of even single-based substitution in RNAs, in straightforward, simple, and cost effective manner.It is anticipated that this analytical tool will aid new finding in molecular biology Begin by identifying gene fragments with different melting profiles and generate predicted melting curves using the Umelt HETS web-based tool. Design three pairs of 300 to 324 base pair gene fragments for each target gene. Select the non-edited or edited pair that shows the maximum difference between the melting regions on the helicity axis for further analysis.For micro-TGGE analysis, synthesize the selected gene fragment by PCR amplification. Design the forward and reverse primers using the DNA dynamo software, and verify the primer sequence using the NCBI Primer-BLAST tool. Extract total RNA from the source of edited and non-edited genes using standard methods.Synthesize the corresponding cDNA, prepare 20 microliters of the PCR reaction mixture, and set up the PCR as described in the text manuscript. Then, mix six microliters of the PCR product with three microliters of 6x gel loading dye in a 250 microliter tube, and make up the total volume to 12 microliters with sterile water. Store the PCR product at 25 degrees Celsius until further use.To assemble the gel cassettes, sandwich the top gel plate between the other two plates in the gel cassette holder for gel polymerization. To prepare the poly acrylamide gel, add 7.2 grams of urea to a 50 milliliter tube and dissolve in 10 milliliters of sterile water. Heat the sample in a microwave for 20 to 30 seconds.And let it cool to room temperature. Add three milliliters of 5x TBE buffer, 2.25 milliliters of acrylamide bis, 75 microliters of 10x ammonium persulfate, and 15 microliters of TEMED to the solution. Pour the gel solution into the gel cassette holder slowly at a tilted angle to avoid air bubbles.After around 30 minutes, disassemble the gel cassettes and clean them. Use the micro-TGGE apparatus with a temperature gradient set perpendicular to the direction of DNA migration. Soak the upper and lower electrophoresis buffer pads in two milliliters of 1x TBE buffer.Place the gel cassette in the horizontal electrophoresis chamber unit, and position the upper and lower buffer pads. Then load 10 microliters of the PCR product into the middle well, and one microliter of the PCR product into each side well. Wait for a minute, connect the power unit and supply 100 volts for 12 minutes at a linear temperature gradient of 15 to 65 degrees Celsius.After the run has completed. take out the cassette and remove the upper glass cover. Pour 300 microliters of 10x SYBR Gold stain onto the gel.Visualize the melting profiles using the blue LED flashlight installed in the palm-sized electrophoretic device. Repeat every electrophoretic experiment thrice to confirm the reproducibility of the data. Download and open the micro-TGGE analyzer software.Open the JPEG file containing the gel image. Click on the frame"button and select the appropriate frame of the gel image. Click on the coordinate correction"button and add two reference points.Click on the addition of feature points"button and add sample points. Then, save the processed gel image data in micro-TGGE format. Click on the sample button and select the search for simple points"option to compare two or more images.For the C-to-U RNA editing type, the non-edited sample with the original cBase, showed a longer melting pattern at the strand end melting point than the edited sample with the modified U base. For the A-to-I RNA editing type, the edited sample with the modified G base, displayed a longer melting pattern at the strand-end melting point than the non-edited sample with the original A base. For the reverse U-to-C RNA editing type, the edited sample in the first gene with the modified C base showed a longer melting pattern between the strand's initial and end melting points than the non-edited sample with the original U base.However, a similar pattern was not observed for the other gene. The pattern similarity scores of the C-to-U and A-to-I RNA editing types were lower than those of the two U-to-C RNA editing types. This difference was likely related to the respective locations of the editing sites.The uMelt HETS analysis showed the C-to-U modification would shift the melting curve to the left along the temperature axis. The pattern similarity scores calculated from micro-TGGE analyses of the non-edited and the three edited fragments were consistent with the results predicted using uMelt. Optimization of the target gene fragment is critical for clear differentiation between the edited and non-edited reason.And this process has been simplified in the current protocol.

a-nonsequencing-approach-for-the-rapid-detection-of-rna-editing

Research

JoVE Journal

Bioengineering

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.