A Nonsequencing Approach for the Rapid Detection of RNA Editing

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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; ADAR1

Dyskusje

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...

Materiały

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