Background priming during reverse transcription by oligo(dT) carried over from mRNA isolation.

The isolation of mRNA from total RNA is the first step in many experimental protocols such as the construction of cDNA libraries. Typically, mRNA isolation is accomplished using commercially available kits that provide some form of free or immobilized oligo(dT) to capture RNA with a poly(A) tail. The oligo(dT)/mRNA complex can be captured by different methods such as by streptavidin-biotin interaction on a magnetic particle or by physical retention on a cellulose matrix. We observed that some of our plant mRNA preparations obtained using various mRNA isolation methods could be reverse-transcribed in the absence of any added primer. The yield and size distribution of the first-strand cDNA population synthesized without added primer were similar to that synthesized in the presence of added oligo(dT). Similar background priming was previously reported in total RNA preparations and attributed to the presence of small nucleolar RNAs that primed randomly (1). Likewise, efficient cDNA synthesis in the absence of oligo(dT) primer is thought to occur in mRNA preparations because of contamination with fragments of DNA or RNA that can bind to mRNA at random sites and serve as a primer, in which case, a reselection for poly(A) RNA is recommended (2). In our experiments, however, isolating poly(A) RNA using two rounds of purification did not prevent first-strand synthesis in the absence of a primer. The size distribution of the first-strand cDNAs obtained from these experiments suggested that the endogenous priming was occurring at the 3′ end of the mRNA and not randomly. To demonstrate that the first-strand synthesis started from the poly(A) tail, we attempted to amplify two long fulllength cDNAs, the Arabidopsis thaliana ferredoxin-dependant glutamate synthase, (4947 bp, GenBank® accession no. Y09667) and the A. thaliana histone acetyltransferase 13 (5811 bp, GenBank accession no. AF510669), using an oligo(dT) primer in combination with a specific 5′-end primer. Both full-length cDNAs were successfully amplified by RT-PCR from the purified first-strand population generated without the addition of oligo(dT) primer (data not shown). This indicates that at least some background priming of cDNA had originated at the poly(A) tail. A possible explanation for this background priming is the presence of exogenous oligo(dT) carried over from the mRNA isolation process. To test this hypothesis, we compared A. thaliana mRNA preparations from four different methods for the presence of oligo(dT). In method 1, 0.9 mg total RNA and 400 pmol of a 5′-biotinylated oligonucleotide GAGAt25 [(gaga)3 ctc gag t25] were mixed to a volume of 1.16 mL and then heat denatured at 65°C for 10 min. Thirty microliters of 20× SSC were added, and the mixture was left at room temperature for 30 min. Streptavidin-magnetic particles (150 μL in 5× SSC; Roche Diagnostics, Laval, QC, Canada) were added, and the mixture was incubated for 10 min at room temperature before capturing the particles using a magnetic stand. The particles were washed three times with 1 mL 0.1× SSC, and the mRNA was eluted twice with 100 μL water preheated to 65°C. In method 2, 0.2 mg RNA and 150 pmol biotinylated oligo(dT) (Promega, Madison, WI, USA) in 0.5 mL water were heat-denatured at 65°C for 10 min. Thirteen microliters of 20× SSC were added, and the mixture was left at room temperature for 10 min before adding 0.6 mL in 5× SSC streptavidinmagnetic particles and incubating at room temperature for 10 min. The particles were captured and then washed four times with 0.3 mL 0.1× SSC, and the mRNA was eluted twice with 125 μL water preheated to 65°C. In method 3, 0.2 mg total RNA in 200 μL water was heat-denatured for 2 min at 65°C before being mixed with the equivalent of 400 μL oligo(dT)25linked paramagnetic polymer particles (Dynal, Lake Success, NY, USA) that were concentrated to 200 μL in 20 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 2 mM EDTA. The mixture was gently agitated for 5 min at room temperature. The particles were captured and washed twice with a buffer of 10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA before eluting the mRNA twice with 50 μL water preheated to 80°C. In method 4, 0.2 mg RNA in 450 μL water was heated for 5 min at 65°C and then mixed with 100 mg oligo(dT)25 cellulose (New England Biolabs, Mississauga, ON, Canada) and 50 μL 5 M NaCl. After 5 min incubation at room temperature, the cellulose was pelleted, and the supernatant was heat-denatured and incubated again with the same cellulose for another cycle of selection. The cellulose was washed four times with 400 μL wash buffer (10 mM TrisHCl, pH 7.5, 0.5 M NaCl, 1 mM EDTA), once with 400 μL low-salt buffer (10 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mM EDTA), and the mRNA was eluted twice with 200 μL 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, preheated to 70°C. The mRNA preparations obtained with the four different methods were used to carry out reverse transcription reactions with and without the addition of exogenous primer. First-strand cDNA was prepared from 50 ng mRNA in 20 μL volume using 100 U Benchmarks