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>
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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.

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