We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.
Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5'-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5' ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.
Primer extension1 is a molecular method to determine the 5’ ends of specific RNA molecules up to a one base resolution. The advantage to other methods such as 5’-RACE (rapid amplification of cDNA ends) is the fast turnaround time and the ability to easily analyze a mixture of different lengths of RNA molecules.
This method works by subjecting RNA molecules to reverse transcription reactions using specific fluorescent primers, generating cDNA fragments of certain lengths. These cDNA molecules are run alongside traditional Sanger sequencing reactions2 on denaturing polyacrylamide gels and can be detected by their fluorescence due to the use of fluorescently labeled primers. The lengths of the cDNA fragments are then assessed by comparison to the sequencing ladder, allowing the mapping of the 5’ RNA ends.
Traditionally, primer extension reactions are used in conjunction with radioactive isotopes to detect cDNA molecules on X-ray films. Due to health hazards, waste disposal issues and ease of handling, newer protocols utilize fluorescence for the detection of the primer extension with automated sequencers, albeit their sensitivity is slightly lower. Using fluorescently labeled primers, the recurring radio-labeling procedure can be omitted, as fluorescent primers are stable for a long time (more than a year in our hands).
The method we describe here utilizes an automated gel sequencer, but with slight modifications, capillary sequencers can be also used for the cDNA separation and detection3. The parallel nature of gel analysis makes it possible to detect even a small amount of RNA cleavage or processing. Another advantage is the high resolution of this method, as terminal cleavage or processing of even one base can be detected.
In regard to the detection of RNA cleavage or processing, typically two different types of primer extensions are distinguished. In one case, the enzymatic treatment is done in vitro using purified RNA and purified enzyme, whereas in the other case, the processing is done in vivo and the resulting RNA is purified. In both cases the RNA is subjected to a primer extension carried out in vitro, however, depending on the source of RNA, the method is either called an in vitro or in vivo primer extension. In the protocol we present here, we focus solely on the in vivo primer extension, because of ease of use (no purified proteins necessary) and the possibility to determine transcriptional starting points and processing at the same time. However, in vitro primer extensions are in principle set up the same way and this protocol can serve as a starting point.
The method illustrated here can be applied to many bacterial species as long as they are amenable to high purity and high-yield preparation of nucleic acids.
The research in our lab focuses on the regulatory scope of toxin-antitoxin (TA-) systems4,5, a field in which the primer extension method is extensively used. TA-systems are small genetic elements present in prokaryotic genomes that consist of a stable and endogenously active toxic protein and a mostly unstable protein or RNA antitoxin that counteracts toxicity6,7. Toxin activity is sometimes exerted by inhibition of replication, cell wall synthesis or other mechanisms, but most often by RNase activity8,9. Typically, RNase specificity is determined by conducting different tests, one of which is the primer extension method. Primer extension reactions are well suited for this application, as a mixture of cleaved and full length fragments can be simultaneously analyzed to determine their 5’ ends. Using a mix of in vitro and in vivo primer extensions, the specific toxin RNase cleavage, e.g., sequence specificity can be determined10-13.
Figure 1. Overview of primer extension procedure. Bacterial cultures are incubated and treated according to the experimental needs. Total RNA is extracted from the cells, treated with DNase I to remove DNA traces and subjected to a reverse transcription reaction using target specific fluorescent DNA primers yielding cDNA. Genomic DNA or plasmids are extracted and subsequently used for fluorescent Sanger sequencing reactions for size comparison with the cDNA fragments. Primer extension products are run alongside Sanger sequencing products on a denaturing urea polyacrylamide gel and analyzed with an automated laser and microscope. The sequencing base that lines up with the cDNA band is the last base of the 5’ cDNA end (blue arrow). More information in Fekete, et al. 3 Please click here to view a larger version of this figure.
An overview of the whole primer extension procedure can be found in Figure 1. Briefly, bacterial cells are cultured, harvested, the cell pellet lysed and the RNA extracted. Purified RNA is then treated with DNase I to remove traces of DNA molecules which could act as templates for the reverse transcriptase. Specific fluorescent primers are added to the RNA, hybridized to the region of interest and subsequently reverse transcribed, resulting in single stranded complementary DNA (cDNA). A sequencing ladder is created by traditional Sanger sequencing employing fluorescent primers and separated on a denaturing polyacrylamide gel alongside of the primer extension cDNA fragments. The resulting gel is analyzed by comparing the fluorescent bands, allowing the identification of the 5’ ends of interest. Transcriptional starting points and processing sites are then assessed individually by sequence comparisons.
1. High Yield RNA Preparation
2. Primer Extension Reaction
3. Preparation of the Sequencing Ladder
NOTE: The sequencing ladder reaction requires either moderate amounts of plasmids or high amounts of genomic DNA. Whenever possible, the use of plasmids in the sequence reaction is recommended due to the ease of isolation and high signal intensity. In other cases, we routinely use a method adopted from Marmur5,14 to prepare genomic DNA from E. coli and S. aureus cells without the need to use phenol. In principal any method that yields high amounts and purity of genomic DNA can be used.
Figure 2. Instruction on how to create a DNA fishing rod. Hold the tip of a glass Pasteur pipette into the flame of a Bunsen burner. This causes the glass to start melting after several seconds, creating a small hook at the end. Quickly remove from the flame and let cool for 1 min.
4. Gel Setup and Apparatus Run
NOTE: Detailed information on how the sequencing gel apparatus is assembled, the gel is prepared and how the gel is run can be found in the manufacturer protocol.
Figure 3. Exploded view of the gel electrophoresis glass plates. Glass plates should be used directionally. Take care to face the inner side of the glass plates inwards and the outer side outwards.
Figure 4. View of an assembled gel apparatus. After injecting the gel solution, the pocket spacer is placed in the solution between the glass plates. The casting plate is then slid between the front glass plate and the gel rails and secured by fastening the rail knobs.
Figure 5. Close-up view of gel with shark tooth comb. Sample (purple) is applied in between the shark teeth.
As depicted in Figure 6, a primer extension reaction can be used to determine the transcriptional starting points of transcripts of interest and can help to deduce promoter regions (typically identified by -10 and -35 elements). The topmost (longest) cDNA fragment represents the 5’ end of the mRNA and thus can be easily mapped when compared to the sequencing ladder.
Figure 6. Representative result of a primer extension reaction. On the left side a full in vivo primer extension gel from E. coli with various plasmids is shown. Individual areas of interest are enlarged. In the top part (A), the determination of the ompA transcriptional starting point is depicted. The reverse transcription stops at the 5’ end of the mRNA and thus creates a band of the full length RNA (indicated by an arrow). By aligning the cDNA band with the sequencing ladder, the 5’ end of the mRNA can be determined as shown in the accompanying sequence. In the lower part (B), the cleavage of the ompA transcript by the YoeB-seq2 RNase is shown. Lanes 1 – 3 represent samples in which the toxin YoeB-seq2 is missing or inactive, whereas lanes 4 – 5 represent samples with an active RNase, coinciding with the absence and presence of cDNA products. Two main cleavage products are created as indicated by the arrows. The ddNTP used in each lane and the corresponding RNA base of the sequencing ladder is labeled. Transcript parts are indicated by blue font, promoter elements and AUG start codon by magenta font. For more information; see the original research article from Nolle et al., Microbiology 159, 1575-1585 (2013). Please click here to view a larger version of this figure.
In the example shown in Figure 6, the TSP of the ompA mRNA was determined to be a G base (marked with an arrow in the sequence below the gel). This is consistent with the ompA TSP published before15. The -35 and -10 elements of the promoter can be deduced to be TTGTAA and TAGACT16 as the motifs only differ two bases each from their respective consensus sequences (TTGACA and TATAAT respectively).
In contrast to other methods such as 5’ RACE, primer extension reactions can be used to accurately determine and quantify the cleavage of RNA molecules. Cleavage of RNA molecules creates free 5’ ends, which can be simultaneously detected as cDNA bands in the gel. Identifying several cleavage products of one mRNA is more difficult in 5’ RACE experiments, since several PCR products must be sequenced to obtain cleavage products that only represent a small fraction of the total (unprocessed) RNA bulk.
In the lower part of Figure 6, the RNA mapping of the cleavage by an RNase is depicted. As previously described5, the RNase YoeB-seq2, part of a TA-system from S. equorum17, cleaves mRNAs close to the start codon. This cleavage can be inhibited by the cognate anti-toxin YefM-seq2. When the RNase YoeB is not present or inactive (lanes 1 – 3), no primer extension products are formed close to the start codon. Whenever RNase activity is present (lanes 4 & 5), two strong bands shortly downstream of the start codon appear. Using this approach, cleavage patterns can be easily identified.
Figure 7 shows a failed primer extension experiment. Due to excess RNA in the reverse transcription reaction, the generated amount of cDNA creates such a strong signal, that individual cDNA bands cannot be distinguished. This makes 5’ RNA end determination impossible.
When using highly expressed RNAs in the primer extension reaction, such as a ribosomal RNA (as depicted in Figure 7), the risk of over-exposed areas increases. Therefore, the amount of total template RNA must be adjusted to the abundance of the RNA of interest. In contrast, when transcripts of interest only constitute a small amount of the total RNA, the amount in the reverse transcription reaction must be increased, otherwise the signals will be too faint (not shown). This also applies to the Sanger sequencing reaction, as multicopy templates per cell (such as rRNA genes or genes encoded on plasmids) result in much stronger signals.
Figure 7. Representative result of a failed primer extension from S. aureus. 16S RNA is highly abundant in bacterial cells. This leads to strong reverse transcription signals when moderate amounts of total RNA are used. In the case depicted here, the strong cDNA bands mask the exact 5’ end and therefore prevent 5’ mapping. In addition, ribosomal RNAs are highly structured and therefore can terminate reverse transcription prematurely, producing short cDNA products that do not represent full length fragments. In this case increasing reverse transcription temperature, together with a heat resistant reverse transcriptase can make it easier to synthesize past secondary structures. Due to multiple copies of the sequence in the genomic DNA, the Sanger sequencing ladder is stronger than for single copy genes. To improve the gel picture, RNA and DNA amounts for the reverse transcription and Sanger reaction should be reduced and/or less product applied to the gel. Please click here to view a larger version of this figure.
Table 1: DNase I digestion of RNA.
Substance | Amount |
RNA from step above | 70 – 100 µg |
10x DNase I Buffer (100 mM Tris, pH 7.5, 25 mM MgCl2, 5 mM CaCl2) | 50 µl |
DNase I, RNase free (2 U/µl) | 10.0 µl (up to 10 µg of RNA per 1 µl of DNase I) |
Bring volume up to 500 µl with ddH2O (DEPC treated) |
Reaction volume and amount of DNase I can be scaled up or down depending on the amount of RNA used.
Table 2: RNA-Primer hybridization.
Compound | Amount for one reaction |
RNA | 5 – 15 µg |
Fluorescently labeled primer | 2 pmol |
Bring volume of to 6 µl with ddH2O (DEPC treated) |
Set up one reaction per RNA sample and primer.
Table 3: Primer extension master mix.
Compound | Amount for one reaction |
ddH2O (DEPC treated) | 1.3 µl |
AMV RT Buffer (10x) | 1.0 µl |
dNTPs (10 mM, RNase free) | 1.0 µl |
RNase Inhibitor | 0.2 µl |
AMV Reverse Transcriptase (RT) (20 – 25 U/µl) | 0.5 µl |
Scale up to the number of reactions needed.
Table 4: 10x TBE recipe.
Substance | Amount |
Tris Base | 107.8 g |
Boric Acid | 55.0 g |
EDTA | 7.4 g |
Bring volume up to 1,000 ml with ultrapure ddH2O and filter to remove dust and lint |
Filter to remove dust and lint and store at 4°C.
Table 5: Sequencing gel recipe.
Compound | Amount |
Urea | 10.5 g |
ddH2O (MilliQ) | 13.0 ml |
10x TBE | 2.5 ml |
XL Rapid gel solution | 5.0 ml |
TEMED (N,N,N′,N′-Tetramethylethane-1,2-diamine) | 25 µl |
APS (Ammonium persulfate, 10%) | 175 µl |
Process gel solution quickly after the addition of TEMED and APS.
Fluorescent primer extension is a simple and rapid method for determining the 5’ ends of RNAs, either for TSP- or secondary RNA processing identification. Due to the use of fluorescent primers, the reactions can be set up and run without additional security precautions (unlike in case of radioactively labeled primers). As the samples are detected by fluorescence, they can be imaged while the electrophoresis is in progress which allows rapid analysis in comparison to radioactive methods where X-ray films are commonly used.
In general, the quality of the primer extension reaction is strongly dependent on the grade and binding capabilities of the primer. If the binding site is chosen too close to the area of interest, primer smears may mask the signal, whereas a binding site too far away (>300 bp) from the 5’ end may result in a poor signal.
The fluorescent dye must be covalently linked to the 5’ end of the custom DNA oligonucleotide during synthesis and the oligonucleotide should be purified by HPLC to prevent interference with the reverse transcription reaction by residual salts. The oligonucleotides with appropriate dye modification (see reagents list table for more information on compatible dyes) can be ordered from most oligonucleotide synthesis companies and should be stored in the dark. Unfortunately, we are unaware of any enzymatic techniques to attach the dye to previously existing oligonucleotides, as is possible with radiolabelled nucleotides.
In the sequencing system described here, two different fluorescent dyes can be used to simultaneously detect two samples, as their excitation (about 700 and 800 nm) and emission spectra are distinct. Apart from the original manufacturer dyes, other dyes such as indicated in the reagents list can be used to produce excellent results.
Another important factor for primer extension reactions is the RNA quality and amount. Care should be taken to remove DNA contaminants, as reverse transcriptases such as the AMV RT can use DNA as a template18.
As shown in Figure 7, the detected signal strength of bands in the gel run is dependent on the RNA amount used in the reverse transcription reaction. It is therefore essential to adjust the amount of total RNA, depending on the proportional amount of the RNA of interest present in the sample. The low sensitivity of this method is also one of its disadvantages, as low expressed RNAs can be difficult to detect. If no signals can be detected at all, the amount of total RNA can be increased or the RNA of interest can be artificially overexpressed from a plasmid.
Gel detection sensitivity for fluorescence based primer extension is about factor ten lower than 32P or 33P radioisotope based primer extension19. However, this disadvantage can be compensated by adjusting the amount of cDNA loaded on to the gel or by increasing the amount of RNA template used in the reverse transcription reaction. In most cases, sensitivity is high enough for satisfactory results4,5. Sensitivities similar to radioactive primer extensions have been reported when using fluorescent primers in combination with a capillary sequencer3, with the advantage of short exposure times.
Costs comparisons between fluorescence and radioactivity based primer extensions are difficult, as they depend on several factors such as available machines, primers, ladder reactions, availability and maintenance of a laboratory for work with radioactive substances, disposal of radioactive wastes, training and individual health risks. Fluorescent primers are about five to ten times more expensive than non-labelled standard primers (20 bp). However these primers can be used for at least a year (more likely several years) compared to 32P labelled primers, which have a much shorter half-life. Re-labelling of primers is time consuming and costly, due to the need for new radioactive material in frequent intervals. If the same set of primers is used throughout a long time period, fluorescent primers are cheaper than regular, radioactively labelled primers. The main cost point will however be the fluorescent sequencer or imager and purchasing this apparatus solely for the purpose of primer extensions might not be cost-effective. The method described here is rather interesting for groups who are in possession of or have access to such a machine.
If an automated gel sequencer is not available, other methods for fluorescence detection can be used as well. In this case, the gel can be run in a standard electrophoresis apparatus, dried if necessary and then transferred to a fluorescent imager (models are available which are similar to a flatbed scanners). Although the advantage of visualizing the gel during the run is lost, the use of radioactive isotopes can be avoided this way, an important advantage for the experimenter. In addition, if available, a capillary sequencer can be used for separation and real-time detection, which can help to increase sensitivity.
Various methods have been published on how to use fluorescent primer extension with automated gel sequencing machines or capillary sequencers, however these methods often require a cDNA precipitation step to concentrate the sample (and remove impurities)19. In the method presented here, DNA precipitation is not necessary thus reducing the preparation time. More importantly however, omitting this step makes it possible to semi-quantitatively determine the amount of RNA molecules in the sample as the inconsistencies of the precipitation procedure can be eliminated.
It should be noted, that although the 5’ ends of RNA molecules can be mapped in primer extension reactions, processed and primary ends (transcriptional starting points) cannot be readily distinguished. To circumvent these restrictions, meticulous planning of the experiments with appropriate controls is essential. Obvious promoter sequences can indicate the presence of a transcriptional starting point and mutating or deleting promoter elements should then abolish cDNA bands. On the other hand, if such sequences are missing or specific consensus sequences are present, the bands may be caused by processing of the RNA. If the processing enzyme is known and can be purified, in vitro primer extensions can bring clarification, as transcription and processing can be separated. In addition, other methods such as 5’ RACE (including enzymatic enrichment of unprocessed RNA molecules) can complement primer extensions to distinguish transcriptional start sites from RNA processing.
The primer extension method is often compared to other methods such as 5’ RACE and S1 nuclease protection assays and thus its usefulness is sometimes questioned. RNAseq techniques in combination with next generation sequencing for example can help to determine TSPs and processing sites of many of RNAs in parallel, however the financial burden and bioinformatical work required make it rather uneconomic for single RNAs of interest. 5’-RACE on the other hand is cheaper and the results are easier to analyze, but if RNAs are processed in multiple ways or several transcriptional starting points are present, the product must be cloned and a large amount of candidates need to be sequenced to obtain a representative view of the RNAs of interest.
Therefore, in spite of new methods having arisen over the years, even today primer extensions have their raison d’être due to the ease of use, low cost and short turnaround time, which is especially true for the fluorescence based method presented here.
The authors have nothing to disclose.
We thank Anne Wochele for her assistance in the laboratory and Vera Augsburger for help with the automated gel sequencer. We thank the Deutsche Forschungsgemeinschaft for funding by grants BE4038/2 and BE4038/5 within the “priority programmes” SPP1316 and SPP1617.
Name | Supplier | Catalog Number(s) | Comment |
AMV Reverse Transcriptase (20-25 U/µl) | NEB / Roche | NEB: M0277-T / Roche: 10109118001 | |
DNase I (RNase free) | Ambion (life technologies) | AM2222 | |
FastPrep-24 Instrument | MPBio | 116004500 | |
Fluorescently labeled primers | Biomers | n/a | 5’ DY-681 modification of “ordinary” DNA oligonucleotides. Compatible dyes such as the LICOR IRDye 700/800 are also available from other suppliers such as IDTdna. |
Li-Cor 4200 Sequencer incl. ImagIR Data collection software | Li-Cor | Product discontinued | |
NanoDrop 2000 | Thermo Scientific | ||
Nuclease free water | Ambion (life technologies) | AM9915G | |
Plasmid mini preparation kit | QIAGEN | 12125 | |
RapidGel-XL-40% Concentrate | USB | US75863 | |
RNA STORAGE BUFFER | Ambion (life technologies) | AM7000 | |
Roti-Aqua-P/C/I | Carl Roth | X985.3 | Alternative: “Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)” from Ambion (AM9720) |
SUPERase•In RNase Inhibitor | Ambion (life technologies) | AM2696 | |
Thermo Sequenase fluorescently labelled primer cycle sequencing kit with 7-deaza-dGTP | GE Healthcare | RPN2538 | |
TRIzol reagent | life technologies | 15596-026 | |
Zirconia/Silica Beads 0.1 mm | BioSpec | 11079101z |