A Nonsequencing Approach for the Rapid Detection of RNA Editing

Summary

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 (µTGGE) as a simple, quick, and portable method of detecting RNA editing.

Abstract

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 (µTGGE) 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 µTGGE analyses. We used the µTGGE-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.

Introduction

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) approaches^1,2,3,4,5,6. 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 detection^7,8. 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 gradient^9,10. 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 (µTGGE), 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 µTGGE 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.

Protocol

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.

1. To identify gene fragments with different melting profiles, representing edited versus nonedited regions, generate predicted melting curves using the uMelt HETS web-based tool, an extension of the original uMelt software¹³.
   NOTE: This tool predicts the shapes of melting curves for heteroduplex and homoduplex products.
2. For each target gene, design three pairs of gene fragments (300-324 bp in length). Each pair comprises of a fragment with an edited site and a corresponding wild-type fragment with a nonedited site, located at either the 5'-terminal end, middle position, or 3'-terminal end.
3. Select the nonedited/edited pair that showed the maximum difference between the melting regions on the helicity axis for further analysis. For µTGGE analysis, synthesize the selected gene fragment by PCR amplification, as described in section 2 below.
4. Design the forward and reverse primers using DNADynamo software (https://www.bluetractorsoftware.com/DynamoDemo.htm) and verify using the NCBI Primer-BLAST tool.
5. Dilute the primers in TE buffer (10 mM Tris-HCl containing 1 mM EDTA·Na₂) and store them at a concentration of 100 pmol/µL. Prior to use, dilute each primer set to a concentration of 10 pmol/µL using distilled water.

2. RNA extraction and RT-PCR amplification of the target fragment

1. RNA extraction
   1. Extract total RNA from the source of edited and nonedited genes using standard methods, such as TRIzol extraction¹⁴ or commercially available kits.
   2. Perform the synthesis of the corresponding cDNA using a standard RT-PCR protocol as described in step 2.2.
2. Reverse transcription
   1. Add 1 µL of oligo(dT) primer, 1 µL of 10 mM dNTP mix, 10 µL of total RNA, and 10 µL of sterile distilled water to a nuclease-free microcentrifuge tube.
   2. Heat the mixture at 65 °C for 5 min and then incubate on ice for at least 1 min.
   3. Collect the contents of the tube by centrifugation at maximum speed (12,000 x g) for 2-3 s, and add 4 µL of 5x ReverTra Ace buffer, 1 µL of 0.1 M DTT, and 1 µL of M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase (200 U/µL, Table of Materials).
   4. Mix by pipetting up and down gently. If using random primers, incubate the tube at 25 °C for 5 min.
   5. Incubate the tube at 55 °C for 60 min and then inactivate the reaction by heating at 70 °C for 15 min, in a digital dry bath/block heater.
   6. Measure the concentration of cDNA using a spectrophotometer and store it at -25 °C.
3. PCR amplification and confirmatory sequencing of products
   1. Add the following reagents to a nuclease-free microcentrifuge tube: 4 µL of 5x Taq Polymerase Buffer, 2 µL of 2 mM dNTP mix, 2 µL of 25 mM MgCl₂, 1.6 µL of the primer set (forward and reverse; each 10 mM), 2 µL of cDNA template, 0.1 µL of Taq polymerase (2.5 U/µL), and 8.3 µL of sterile distilled water (final volume, 20 µL).
   2. Perform PCR with the following cycling conditions: initial denaturation at 95 °C for 2 min; 35 cycles of denaturation at 95 °C for 2 min, annealing at a suitable temperature (depending on the specific primer set used) for 30 s, and extension at 72 °C for 2 min; and then a final extension at 72 °C for 7 min.
   3. To purify the PCR products, add 2 U of Exonuclease I and 0.5 U of shrimp alkaline phosphatase (ExoSAP). Incubate the tube in a thermal cycler at 37 °C for 1 h, followed by 80 °C for 15 min, and then maintain at 4 °C until the next step.
   4. For confirmatory sequencing, add 11.5 µL of sterile distilled water, 2.5 µL of forward or reverse primer, and 1 µL of PCR products (with ExoSAP) to a new microcentrifuge tube.
      ​NOTE: Use DNA sequencing services (e.g., Eurofins Genomics) for sequencing analysis.
   5. To validate the specificity of the PCR products, perform sequencing with both forward and reverse primers. The primer pairs used in the representative analysis are given in Table 1.
   6. Dilute the purified PCR products to a concentration of 200 ng/µL with deionized water. Next, mix 6 µL of the purified product with 3 µL of 6x gel loading dye in a 250 µL tube and raise the total volume to 12 µL with sterile water. Store the PCR product (at -25 °C) until ready to proceed with µTGGE as described below.

3. µTGGE analysis

NOTE: The µTGGE analysis is performed using a miniaturized and economic system. An overview of the complete system, including the gel cassettes, gel cassette holder, horizontal gel electrophoresis platform, power supply, and gel imaging system, is shown in Figure 2.

1. Assembly of the gel cassettes
   1. The gel cassette used for µTGGE analysis is shown in Figure 2A. The design consists of three 1 inch gel plates: a bottom gel plate, a top gel plate, and a lane-former plate. Sandwich the top gel plate between the other two plates and assemble in the gel cassette holder for gel polymerization.
2. Polyacrylamide gel preparation
   1. Add 7.2 g of urea to a 50 mL tube and dissolve in 10 mL of sterile water. Heat the sample in a microwave for 20-30 s and then bring it to room temperature (RT).
   2. Add 3 mL of 5x Tris/borate/EDTA (TBE) buffer (Table of Materials), 2.25 mL of 40% (w/v) acrylamide/bis (19:1), 75 µL of 10x ammonium persulfate, and 15 µL of tetramethylethylenediamine to the solution.
   3. Immediately pour the gel solution into the gel cassette holder slowly, avoiding the formation of air bubbles.
3. Generation of melting profiles using the µTGGE unit
   1. Use the miniaturized, economical version of the µTGGE apparatus shown in Figure 2 with a temperature gradient (25-65 °C) set perpendicular to the direction of DNA migration.
   2. Soak the upper and lower electrophoresis buffer pads (0.5 cm x 2.5 cm) in 2 mL of 1x TBE buffer.
   3. Place the gel cassette in the horizontal electrophoresis chamber unit and position the upper and lower buffer pads accordingly, as shown in Figure 2.
   4. Next, load 10 µL of the PCR product into the middle (longer) well and 1 µL of the PCR product into each side well.
   5. After a 1 min wait, connect the power unit and supply 100 V for 12 min at a linear temperature gradient from 25-65 °C.
   6. After the run has completed, take out the cassette and remove the upper glass cover.
   7. Pour 300 µL of 10x SYBR Gold stain onto the gel. Visualize the melting profiles using the blue LED flashlight installed in the palm-sized electrophoretic device.
      NOTE: A soft file of the gel image should be saved for Pattern Similarity Score (PaSS) calculation.
   8. Repeat every electrophoretic experiment 3x to confirm the reproducibility of the data.

4. PaSS calculations

NOTE: The calculations are performed using µTGGE Analyzer software.

1. Download and open the software.
2. Open the JPEG file containing the gel image. Note that the software will only accept JPEG format files.
3. Click on the Frame button and select the appropriate area (frame) of the gel image.
4. Click on the Coordinate Correction button and add two references points, as shown in Figure 1B.
5. Click on the Addition of Feature Points button and add sample points as shown in Figure 1B. Then save the processed gel image data in µTGGE (*.tgg) format.
6. Click on the Sample button and select the Search for Simple Points option to compare two or more images.

Representative Results

Use of µTGGE 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; ADAR1¹⁶), and the AT2G16586 and AT5G02670 nuclear genes in A. thaliana^17,18 (with U-to-C RNA editing). Single nucleotide differences between the edited and corresponding nonedited samples were confirmed by DNA sequencing. Then a µTGGE analysis was performed to examine the differences between the melting curves of the samples (Figure 3).

For the C-to-U RNA editing type, the nonedited sample with the original C base showed a longer melting pattern at the strand end-melting point than the edited sample with the modified U base (Figure 3A). For the A-to-I(G) RNA editing type, the edited sample with the modified I(G) base displayed a longer melting pattern at the strand end-melting point than the nonedited sample with the original A base (Figure 3B). For the "reverse" U-to-C RNA editing type, two genes were analyzed. For the AT2G16586 gene, the edited sample with the modified C base showed a longer melting pattern between the strand initial-melting and strand end-melting points than the nonedited sample with the original U base (Figure 3C). However, a similar pattern was not observed for the other AT5G02670 gene (Figure 3D). Nonetheless, there was a distinct difference between the nonedited and edited types at the end-melting point. This shows that observing melting profiles clearly is an important step in distinguishing between nonedited and edited types.

Quantitative analysis of µTGGE melting patterns representing RNA editing events
Next, we calculated PaSS values¹¹ to evaluate the reproducibility of the µTGGE-based melting profiles representing the four RNA editing events described above. The PaSS value provides a measure of how closely two melting patterns can be superposed, generating a higher value (maximum: 1) for highly similar melting patterns. Thus, PaSS values for comparisons of nonedited and edited samples are expected to be less than one. As shown in Figure 1B, the feature points of the melting patterns that corresponded to structural transitions from double-stranded to single-stranded DNA were used to calculate PaSS values. To eliminate experimental variables, computer-aided normalization was performed using two internal reference points: reference point #1, representing the position of the sample in double-stranded form (the leftmost lane), and reference point #2, representing the position of the sample in single-stranded form (the rightmost lane). The coordinates of the feature points were normalized to those of the internal reference points and were then used to calculate the PaSS values. Each experiment was repeated three times, and the average value was determined. As expected, the PaSS values of the four edited samples were lower than one (Figure 4). The PaSS values 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 is likely related to the respective locations of the editing sites. Specifically, the C-to-U and A-to-I edited sites were located relatively close to the 5'-terminal ends (at positions 48 and 59 of the ~300 bp fragments, respectively), whereas the U-to-C edited sites were located near the middle of the fragments (at positions 152 and 169). These findings suggest that edited sites located at terminal positions can be detected using µTGGE more easily than those located toward the center of fragments.

Optimization of TGGE melting patterns to identify RNA editing events
As our previous results suggested that the PaSS value may vary depending on the specific position of the RNA editing site, we examined the differences between the melting patterns of a 300 bp fragment of the BFP gene (expressed in HEK293T cells) in which a C-to-U edited site was located close to the 5'-terminal end, close to the 3'-terminal end, or in the center of the fragment (Figure 5A). Prior to µTGGE analysis, the melting patterns of the nonedited fragment and three edited fragments were predicted using the uMelt HETS web-based tool. This analysis showed that the C-to-U modification would be expected to shift the melting curve to the left along the temperature axis (Figure 5B). The PaSS values calculated from µTGGE analyses of the nonedited and the three edited fragments were ordered as follows: 5'-terminal end RNA edit < 3'-terminal end RNA edit < center of 5'- and 3'-terminal ends RNA edit (Figure 5C). Notably, these PaSS values were consistent with the results predicted using uMelt. These findings indicate that nucleotide base differences located at the 5'- or 3'-terminal end result in larger variations between the PaSS values of edited and nonedited genes than nucleotide base differences located more centrally. In addition, these results suggest that prior knowledge of the differences between the melting profiles of edited and nonedited genes can be used as a guide to optimize gene fragments for RNA editing.

Figure 1: The procedure used to identify RNA editing by µTGGE. (A) The types of RNA editing events examined. (B) A schematic illustration of the typical melting profiles of edited and nonedited genes. In µTGGE, a sample migrates through a temperature gradient gel, producing a characteristic curvature. The feature points of the melting pattern are assigned and then processed to calculate a Pattern Similarity Score (PaSS) value. The PaSS calculation is performed as shown in the equation, where the vector P of each feature point is in its corresponding position and the function of temperature and mobility (i.e., vector P = P (T, m)). The superscripts (1) and (2) represent the edited and nonedited genes, respectively. Please click here to view a larger version of this figure.

Figure 2: Illustrations and photographs of the palm-sized gel electrophoresis device. (A) Assembly of the three 1 in gel cassettes. (B) Illustration of the gel cassette holder with the temperature gradient plate. The photograph shows the positions of the upper and lower buffer pads, as well as that of the sample after initial loading. (C) Overview of the complete system, including the power supply, horizontal gel electrophoresis platform, and gel imaging system. Thissystem provides a viable solution for rapid, onsite polyacrylamide gel electrophoresis-based analyses. Please click here to view a larger version of this figure.

Figure 3: TGGE analyses of single nucleotide changes in RNA editing events. Melting profiles for three different RNA editing types were examined using four genes. (A) C-to-U RNA editing in BFP expressed in HEK293T cells. (B) A-to-I(G) RNA editing in EGFP expressed in HEK293T cells. (C) U-to-C RNA editing in the AT2G16586 gene from A. thaliana. (D) U-to-C RNA editing in the AT5G02670 gene from A. thaliana. The locations of the edited sites are highlighted in yellow (with red font), and the primer positions are underlined. The differences between the melting patterns of the edited and nonedited samples are indicated by red circles. Please click here to view a larger version of this figure.

Figure 4: The average PaSS values for the four edited genes examined here. Error bars represent the standard deviation of three replicates. Please click here to view a larger version of this figure.

Figure 5: Position-specific PaSS analysis. (A) The C-to-U type RNA editing site in the BFP gene expressed in HEK293T cells was shifted to the 5'-terminal end, 3'-terminal end, or center of the gene fragment. (B) Theoretical prediction of the melting patterns of the nonedited and three edited fragments shown in (A). The predictions were performed using uMelt. (C) The average PaSSvalues of the edited genes shown in (A). Error bars represent the standard deviation of three replicates. Please click here to view a larger version of this figure.

S.No	RNA editing	Source	Position	Gene ID	Forward Primer		Reverse Primer		Sequence length
1	C-to-U	HEK293T cells	48th	EGFP	AAGCTGACCC TGAAGTTCATC		GCTGTTGTAGT TGTACTCCAGC		324
2	A-to-I	HEK293T cells	59th	EGFP	AGGGCGATGC CACCTACGGCA		CCGTCCTCCT TTAAGTCGA		300
3	U-to-C	Arabidopsis	152th	AT2G16586	GGGCGATGTT ACGCTCGATGA		GTGAAGAGTAA CATGGCGTT		301
4	U-to-C	Arabidopsis	169th	AT5G02670	CCAGTTGGCAG AATCCAGTCA		CTAGCTTCCAC TGTTGAGATTC		300

Table 1: List of genes used in the current protocol

Discussion

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 genes with single-base nucleotide modifications can be differentiated based on changes in the TGGE melting profiles. As it incorporates 1 in gels, the system requires very small amounts of samples and enables rapid detection of differences between sequences. In addition, the protocol described here is very easy to follow for both experts and newcomers to the field. Optimization of the target gene fragment is critical for clear differentiation between edited and nonedited regions, and this process is simplified in the current protocol.

This protocol is compatible with multiplex analyses using fluorescently tagged primers. For quantitative analyses, unknown RNA samples (edited or nonedited) can be tagged with green fluorescence and comigrated with a red fluorescence-tagged reference standard (edited or nonedited) during TGGE analysis.

In addition to demonstrating the ability of the µTGGE method to detect single nucleotide RNA editing events, we also validated the similarity between the experimental result obtained using µTGGE and a theoretical result obtained for the same gene fragment using uMELT. Furthermore, we found that nucleotide base differences located at the 5'- and 3'-terminals of gene fragments produce larger differences in µTGGE melting profiles (i.e., smaller PaSS values) than those located toward the center of the fragments. Although promising, the current protocol may be limited to the analysis and detection of specific types of nucleotide modifications during RNA editing. Further optimization and development of this protocol to analyze other types and/or locations of RNA editing will be performed in our upcoming research.

Materials

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