Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Summary

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.

Abstract

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.

Introduction

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 fluores....

Protocol

1. High Yield RNA Preparation

1. RNA Isolation
   NOTE: High concentrations of total RNA are needed for the primer extension reaction. Spin column kits usually do not yield the amount of RNA needed (~5 - 16 µg in 5 µl volume). Therefore purification using the acid guanidinium thiocyanate-phenol-chloroform extraction method is recommended, outlined below.
   NOTE: Phenol is carcinogenic, toxic and corrosive. Please read the material safety data sheets and use under a fume hood with appropriate protection!
   1. Grow or treat the bacterial cells (S. aureus or E. coli in this example) as desired and harvest by 10 min centrifugation at....

Results

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.

Discussion

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 commonl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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