Excess dNTPs Minimize RNA Hydrolysis during Reverse Transcription

Divalent Mg2+-catalyzed degradation of RNA is a welldocumented phenomenon (1,3) that is not widely recognized in the molecular biology laboratory. At temperatures above 37°C (1) and at pH 7.5 and above in Tris-HCl buffers (3), the rate of RNA breakdown is directly proportional to Mg2+ concentration between 1 and 10 mM. Other buffer systems such as glycine (1) or sodium citrate (3) reduce the detrimental effects of Mg2+. Free Mg2+ (1–3.5 mM) and alkaline pH (8.4) in Tris-HCl buffers are present during cDNA synthesis with murine and avian retroviral reverse transcriptases (8,14), creating conditions under which Mg2+-catalyzed RNA breakdown occurs. One significant difficulty with retroviral reverse transcriptase-catalyzed cDNA synthesis is the tendency of the enzyme to pause at regions of secondary structure in mRNA (5,10,20), resulting in the generation of truncated products. As the result, more thermal stable reverse transcriptases are being sought and used that function at temperatures high enough to denature RNA (18), and protocols are being developed that employ RNA heating steps at high temperatures in the presence of Mg2+ (11). To define the stability of RNA under reaction conditions used to synthesize cDNA, we determined the rate of degradation and half-life of a 7.5-kb cRNA at various temperatures under reaction conditions optimal for Moloney murine leukemia virus (MMLV) reverse transcriptase (8). We found that the rate of Mg2+-catalyzed RNA degradation is quite rapid at higher temperatures and that RNA breakdown can be reduced by the addition of excess dNTPs to chelate free Mg2+. The protocol used to measure RNA breakdown was as follows. RNA (7.5 kb; Invitrogen, Carlsbad, CA, USA) was used unlabeled or labeled at the 3′end with [α-32P]ddATP and yeast poly(A) polymerase (Amersham Biosciences, Piscataway, NJ, USA). The results obtained with labeled and unlabeled RNA were similar. RNA was heated at various temperatures in a 0.5mL tube in a thermal cycler equipped with a heated lid. The heating solution (20 μL) mimicking MMLV reverse transcriptase reaction conditions contained 50 mM Tris-HCl, pH 8.4, 75 mM KCl, 3 mM MgCl2, 500 μM each dCTP, dGTP, dTTP, and dATP, and 10 μg unlabeled or 3′-end-labeled 7.5-kb RNA. At various times, 2-μL aliquots were removed and placed on ice in separate tubes containing 1 μL 100 mM EDTA. Gel loading buffer (30 mM MOPS, pH 7.0, 0.6 mM EDTA, 27 mg/mL sucrose, 2.9 M formaldehyde, 57% formamide, 1.3 mg/mL bromocresol green, and 7 μL 70 μg/mL ethidium bromide) was added, and the RNA was heated at 65°C for 5 min. When testing avian reverse transcriptase reaction conditions, the MgCl2 concentration was increased to 7.5 mM and the concentration of each dNTP was increased to 1 mM in heating mixtures. Heated RNA was subjected to denaturing formaldehyde agarose gel electrophoresis as described previously (7). Gels contained 1.5% agarose, 50 mM MOPS, pH 7.0, 1 mM EDTA, and 2.2 M formaldehyde and were run in 50 mM MOPS (pH 7.0)-1 mM EDTA. A digitized image of ethidium bromidestained, unlabeled RNA was captured during transillumination of gels with short-wave UV light with a Lynx 5000 Gel Imaging System (Applied Imaging, Santa Clara, CA, USA). The system software was used to quantify the fraction of intact 7.5-kb RNA present in a lane of the gel. When labeled RNA was used, the gel was dried and exposed to X-ray film. Using an exposed film as a guide, the intact 7.5-kb RNA and any breakdown products were excised separately and counted in a scintillation counter to determine the fraction of intact RNA. cDNA synthesis with MMLV Hreverse transcriptase (SuperScript II reverse transcriptase; Invitrogen) was carried out in reaction mixtures (20 μL) containing 50 mM Tris-HCl, pH 8.4, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 μM each dATP, dTTP, dGTP, and [α-32P]dCTP (400 cpm/pmol), 1750 U/mL RNase Inhibitor, 62 nM 5.2-kb MAP-4 cRNA with a 3′-poly(A) tail (4), 575 nM p(dT)25-30, and 430 nM MMLV Hreverse transcriptase. In some cases, the concentration of each dNTP was increased to 1 mM. Reaction mixtures for Rous sarcoma virus (RSV) Hreverse transcriptase (80 nM ThermoScript reverse transcriptase; Invitrogen) were the same, except the MgCl2 and individual dNTP concentraBenchmarks