Resistant Ribonuclease Activity in Preparations of Total RNA Extracted from Artiodactyl Brain with GITC

The protocol of Chomczynski and Sacchi (1) constitutes an established method widely used for extracting total RNA from many mammalian species. In general, this method produces goodquality total RNA suitable for various techniques such as Northern blot, RTPCR, or RNase mapping. The combined inhibitory effect of both phenol and guanidium isothiocyanate (GITC) allows inactivation of most ribonuclease activity during RNA isolation. RNA quality is usually tested in the following three ways: visualization of 18S and 28S ribosomal RNA with agarose gel electrophoresis, detection of a particular mRNA expression by RT-PCR, and the RNA A260/280 ratio, which gives information about protein contamination. However, in our experiments, a residual degrading activity in ovine RNA prepared with the Chomczynski and Sacchi method has been detected. This degradation was not observed when RNA was dissolved in water, and the controls described above gave correct results. The degradation occurred at a later stage when RNA was incubated in an enzyme buffer such as DNase I buffer, reverse transcriptase buffer, or in vitro transcription buffer. Brain (hypothalamus and cortex) and liver total RNA were extracted from frozen artiodactyl and rodent tissues (-70°C) using the Chomzynski and Sacchi method (1). Briefly, tissue was homogenized at ambient temperature or at 4°C in a denaturing solution (Solution D) composed of 4 M GITC; 25 mM sodium citrate, pH 7.0; 0.5% sarcosyl; 0.1 M β-mercaptoethanol. Onetenth volume 2 M sodium acetate, pH 4.0, 1 volume phenol, and 0.2 volume choroform:isoamyl alcohol mixture (49:1) were successively added. The mixture was vigorously shaken and left for 15 min on ice. After centrifuging at 11 000× g for 20 min, the aqueous phase was mixed with one volume of isopropanol. The precipitated RNA was pelleted by centrifugation at 11 000× g for 30 min and resuspended in Solution D. After a second centrifugation, the pellet was rinsed with 75% ethanol and dissolved in Milli-Q® water (Millipore S.A., St. Quentin Yvelines, France) (2). Ten micrograms of RNA were treated with proteinase K (50 μg/mL final in 10 mM Tris-HCl, pH 7.5; 5 mM EDTA; 0.5% SDS) for 30 min at 37°C and then subjected to one phenol:chloroform extraction, followed by two chloroform extractions and ethanol precipitation with sodium acetate. RNA was dissolved in Milli-Q water. Ten micrograms of untreated RNA and proteinase K-treated RNA were incubated in either Milli-Q water or enzyme buffer for 30 min at 37°C and recovered by ethanol precipitation. After centrifugation, the pellet was resuspended in a denaturing loading buffer, and RNA was separated on 1% agarose gel containing formaldehyde and visualized with ethidium bromide staining. The electrophoresis on the denaturing gel in Figure 1A showed that the method of Chomczynski and Sacchi leads to the preparation of intact ovine hypothalamus RNA, when in Milli-Q water solution, even after 30 min incubation at 37°C (lanes 1 and 2). The A260/280 ratio was generally within an acceptable range (1.6–1.8), considering the slightly acidic pH of the solution after RNA dissolution in water (3). When incubated in DNase I buffer (33 mM Tris-acetate, pH 7.8, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT; BD Biosciences Clontech, Palo Alto, CA, USA) for 10, 20, and 30 min at 37°C (lanes 3–5), RNA was detected as smearing with total disappearance of the 18S and 28S RNA (lanes 4 and 5). This degradation did not occur when the RNA preparation was first subjected to treatment with proteinase K. Proteinase K-treated RNA retained its integrity when incubated for 30 min at 37°C in DNase I buffer (lane 10). Moreover, this degrading activity disappeared when proteinase K-untreated RNA was incubated in DNase I buffer in the presence of RNase inhibitor (Rnasin®; Promega France, Charbonniéres, France) (lane 6). This may mean that the degradation is due to the action of a ribonuclease co-purified with RNA. The same pattern was observed in preparations of bovine and porcine brain RNA (Table 1), from which it could be concluded that this degrading activity may be found in artiodactyl species. This activity is not restricted to the hypothalamus, since we have also found it in cortex RNA preparations (Table 1). In contrast, we did not observe any degradation in rodent brain RNA, even when incubated in DNase I buffer at 37°C. The migration pattern of rat RNA on denaturing agarose gel showed profiles of intact RNA from the hypothalamus or cortex, incubated in either Milli-Q water or DNase I buffer (Figure 1B, lanes 1–4). The same result was obtained with mouse RNA (data not shown). This showed that the residual activity depicted above is species-specific in the brain. While it is found co-purified with RNA from artiodactyl species, no degradation can be detected in rodent RNA preparation when incubated with DNase I buffer at 37°C (results are summarized in Table 1). In sheep and rodents, we observed no difference between males and females (Table 1). To eliminate the latent degrading activity, two additional phenol:chloroform extractions were performed on the aqueous phase before isopropanol precipitation. This modification, intended to remove some residual proteins from preparations, did not alter RNA integrity when incubated in Milli-Q water (Figure 2A, lane 2). Surprisingly, the degrading activity was resistant to the three successive phenol:chloroform extractions (Figure 2A, lane 3). The degradation was also observed when sheep RNA was incubated in a reverse transcriptase buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2; SuperScript II®; Invitrogen S.A.R.L., Cergy Pontoise, France) and in an in vitro transcription buffer (40 mM Tris-HCl, pH 8.0, 8 mM MgCl2, 2 mM spermidine, 50 mM NaCl, Stratagene, La Jolla, CA, USA) (Figure 2B, lanes 3 and 5). The integrity of proteinase K-treated RNA after incubation in these two buffers demonstrated that there was no exogenous RNase contamination during the experiment (lanes Benchmarks