Isolation of Total RNA and mrna from Plant Tissues

Transcription

1 Promega Notes Magazine Number 54, 1995, p.02 Isolation of Total RNA and mrna from Plant Tissues By: Isabel Murillo, Dora Raventos, Estelle Jaeck, Blanca San Segundo* Centro de Investigacion y Desarrollo de Barcelona (CSIC), Departamento de Genetica Molecular Jordi Girona 18, 08034, Barcelona, SPAIN *Corresponding author: Tel: Fax: The Promega RNAgents Total RNA Isolation System and PolyATtract Systems have been used to obtain total RNA and mrna, respectively, from maize tissues. Modifications of the protocols provided by Promega have been made in order to adapt them to use with the different plant tissues studied in this work. Furthermore, the PolyATtract System 1000 has been used for the direct purification of mrna from leaves and germinating embryos. Yields obtained using these systems were better than standard isolation procedures. The RNAs were of high quality and produced strong hybridization signals in Northern blot analysis. The mrna has been used successfully for construction of a cdna library from germinating maize embryos. Introduction Plant tissue is perhaps one of the most difficult materials from which to isolate RNA. The main problems associated with plant RNA isolation have been attributed to copurification of plant polysaccharides and polyphenolic compounds (1,2). These products bind to RNAs during extraction, resulting in the formation of either a viscous, insoluble material that interferes with the isolation steps, or of adducts completely devoid of biological activity. Qualitative and quantitative differences in composition of plant phenolics and polysaccharides in different plant tissues significantly affect the efficiency of nucleic acid extraction and purification procedures. Consequently, in order to isolate RNA from each new plant material examined, several methods should be tested to select the optimal one. RNA is generally isolated by using detergents (e.g., SDS) and organic denaturants (e.g., guanidine thiocyanate) to inhibit ribonucleases and remove the majority of non-nucleic acid components from the final preparation (3). Promega's RNAgents Total RNA Isolation System combines the use of the strong denaturants guanidine thiocyanate and phenol-chloroform with a reducing agent, betamercaptoethanol, to inhibit RNase activity. Promega's PolyATtract Systems are designed to quickly purify large amounts of undegraded mrna from total RNA or tissue by using streptavidin coupled paramagnetic particles (SA-PMPs) to bind biotinylated oligo (dt):mrna hybrids. The PolyATtract System 1000 combines RNA extraction capabilities with the use of SA-PMPs to isolate mrna directly from tissues. This article summarizes the results obtained for extraction of total RNA and mrnas from different maize tissues using the systems developed by Promega. We have made some modifications to the standard Promega protocols in order to adapt them to the isolation of RNA from the plant tissues employed in this work. Isolation of total RNA from maize tissues The RNAgents Total RNA Isolation System and two alternative methods of RNA purification (4,5) were used to obtain total RNA from various maize tissues (leaves from adult plants, coleoptiles, radicles, and the seed tissues, endosperm and embryo). The protocol provided with the Promega system (6) was followed for the isolation of RNA from all tissues except seed. The extraction of RNA from seeds is difficult due to the abundance of polysaccharides in their tissues. For this reason, it was necessary to modify the Promega protocol. In these cases, the final RNA preparation obtained using the RNAgents System was further treated by subjecting it to two consecutive cycles of heating and centrifugation. The RNA solution was heated for 5 minutes at 65 C, incu-bated for 5 minutes on ice and then centrifuged at 12,000 x g for 5 minutes at room temperature. The supernatant was removed, the pellet resus-pended in RNase-free deionized water and the process repeated. The two supernatants were combined (recoveries of total RNA were 75% and 25%, respectively). The RNA was recovered from the supernatant by using a high-salt precipitation procedure (2 volumes of ethanol and 2M NaCl, final concentration) which leaves the polysaccharides in solution (7). The pellet was then resuspended in RNase-free deionized water and reprecipitated as described in the Promega protocol (2.5 volumes of ethanol and 0.25M sodium acetate, ph 5.0, final concentration). The absence of significant levels of polysaccharides was confirmed by UV spectroscopy (A260/A280 of ). Table 1 summarizes the RNA yields obtained from various tissues using the three methods. The RNAgents Total RNA Isolation System proved to be very simple to use and provided high yields from the tissues extracted. The quality of the RNAs was assessed by agarose gel electrophoresis (Figure 1). The RNA was of high quality, as judged by the appearance of the two ribosomal RNA (rrna) bands (25S and 18S, the major plant cytoplasmic rrnas). The bands observed below the 18S band in lane 3 correspond to chloroplast rrnas. The RNA prepared from several tissues was slightly contaminated with high molecular weight DNA, however, the level of contamination in the samples isolated with the RNAgents System was always lower

2 than with other methods (Figure 1, lanes 1 and 2). Figure Analysis of RNA extracted from maize tissues. Non-denaturing agarose gel analysis (4% agarose in TAE buffer (ph 8.0) containing 0.5µg/ml ethidium bromide) of total RNA prepared from maize tissues using either Method II (see Table 1 and reference 4, Lane 1) or the RNAgents Total RNA Isolation System (reference 6 and text, Lanes 2-6). Lanes: Lane 1, embryo, 6µg; Lane 2, embryo, 10µg; Lane 3, leaves, 3µg; Lane 4, coleoptiles, 3µg; Lane 5, radicles, 3µg; Lane 6, endosperms, 5µg. Table Yields of Total RNA from Maize Tissues Using Different Isolation Methods. RNAgents Method II Method III Tissue Type (mg/g fresh weight) (mg/g fresh weight) (mg/g fresh weight) Embryo Radicle Coleoptile Endosperm 0.20 n.d Leaf RNA extractions using the RNAgents Total RNA Isolation System were performed as described (6), except for the modifications as detailed in the text for seed tissue. RNA isolations using Method II and Method III were as described in references 4 and 5, respectively. Yields are expressed as milligram of RNA/gram of tissue. Maize (Zea mays pure inbred line W64A) was used as the experimental material. Leaves were collected 6 weeks after planting, other tissues were from maize seedlings. Seeds were germinated for 7 days and dissected to obtain radicles, coleoptiles, embryos and endosperms. Tissues were immediately frozen in liquid nitrogen. n.d.: Not determined. Isolation of poly(a)+ RNA from total RNA The definition of poly(a)+ RNA is a practical one, based upon oligo(dt) cellulose chromatography analysis, which specifically selects for molecules containing poly(a) tracts of more than residues. In plants, however, there is a wide variation in the length of poly (A) sequences present at the 3 end of individual mrnas. Chloroplasts of higher plants, for example, have poly(a) sequences which are usually no longer than 20 nucleotides (8). Unless special precautions are taken, such mrnas are not bound by oligo(dt) and are lost in the poly(a)- fraction during purification. Taking into account the nature of plant mrnas, we modified the protocol (9) for Promega's PolyATtract System to obtain better selection of poly(a)+ RNA from total RNA prepared from maize tissues (see above and Figure 1). In these exper-iments, the standard protocol was followed to the point where the biotin-oligo(dt)/mrna hybrids were bound to the Streptavidin Coupled Paramagnetic Particles (SA-PMPs). We reduced the stringency of the subsequent wash step by using 0.2X SSC buffer instead of 0.1X SSC buffer as recommended in the Promega protocol, and increased the number of washes from four to five. An electrophoretic analysis of one of the purifications is presented in Figure 2.

3 Figure 2. Non-denaturing agarose gel analysis of mrna isolated using the PolyATtract System. mrna was isolated from the Maize embryo total RNA sample shown in Figure 1 (Lane 2) using the PolyATtract System (three isolations, each starting with 3.8mg of total RNA, were combined). The SA-PMPs were washed as described (reference 9 and the text) and the RNA remaining in the wash solutions analyzed on a non-denaturing gel as detailed in Figure The RNA remaining in washes 2-5 was precipitated and the entire sample loaded on the gel. For wash 1 a volume equivalent to 5µg of RNA (determined by A260 ) was loaded on the gel. Lanes 1-5: wash steps 1-5. Lane 6: 0.5µg of the eluted mrna fraction. The yields of poly(a)+ RNA were in the range of 0.5-5% of the total RNA, depending on the tissue (Table 2). The yields were consistently better than those obtained using standard oligo(dt) cellulose chromatography. For example, the yield of poly(a)+ RNA obtained by oligo(dt) chromatography of total RNA isolated from germinating maize embryos was approximately 0.25%, compared to 0.47% using the PolyATtract System. Furthermore, in many cases two cycles of oligo(dt) chromatography were necessary to obtain highly purified poly(a)+ RNA, resulting in yields lower than 0.25% (data not shown). Table 2. Yield of Poly(A)+ RNA from Maize Tissues Using the PolyATtract System. Amount of Total Yield of Poly(A)+ Percent of Total Tissue Type RNA RNA RNA Embryo 0.25mg 25µg 0.51% Endosperm 0.52mg 7.0µg 34% Radicle 0.30mg 4.6µg 53% Coleoptile 0.38mg 3.0µg 0.79% Leaf 0mg 5.0µg 0.50% Embryo 3.80mg 18.0µg 0.47% Total RNA was isolated using the RNAgents Total RNA Isolation System as described in Table Poly(A)+ RNA was purified from total RNA as described (9), except for the modifications detailed in the text. Specific mrna were intact, as demonstrated by Northern blot analysis using the cdna for a maize defense gene (PRms) (10) as a probe (Figure 3). This analysis revealed no apparent degradation of either the total RNA or the poly(a)+ RNA fraction obtained from maize embryos using the RNAgents Total RNA Isolation System and PolyATtract System, respectively. Finally, the poly(a)+ RNA isolated from germinating maize embryos using the PolyATtract System was successfully utilized in the construction of a cdna library in the Lambda ZAP Cloning Vector (results not shown). Figure 3. Northern blot analysis of maize embryo total and poly(a)+ RNAs. RNA was isolated using either the RNAgents System (total RNA) or the PolyATtract System (poly(a)+ RNA). RNAs were separated by electrophoresis on a formaldehydecontaining 4% agarose gel and transferred to a Nylon membrane. The blot was probed with a 32P-labeled PRms (Pathogenesis- Related protein) cdna (10) and exposed to X-ray film for 48 hours. Lanes: total RNA, 10µg; mrna, 0.5µg.

4 Isolation of poly(a)+ RNA directly from maize tissue We have also used the Promega PolyATtract System 1000 for the direct isolation of mrna from maize embryos and leaves. The isolations were performed as recommended in the Promega technical manual included with the system (11). Table 3 shows the yields obtained from these tissues using as little as 0.5mg of starting material. As shown in Figure 4, agarose gel electrophoresis suggested that the mrnas isolated directly from embryonic tissue were intact. Similar results were obtained for mrna isolated directly from leaves (data not shown). Table 3. Direct Isolation of Poly(A)+ RNA from Maize Tissues Using the PolyATtract System Yield of Poly(A)+ Tissue Type Amount of Tissue RNA Embryo 0.5mg 5.8µg Leaf 0.5mg 2.5µg Poly(A)+ RNA was isolated as described in Table 1 and reference 1 Figure 4. Gel electrophoresis analysis of mrna directly isolated from maize tissues. RNA was isolated from maize embryos using either the PolyATtract System 1000 or the RNAgents Total RNA Isolation System and subjected to agarose gel electrophoresis as described in Figure Lanes: mrna, 0.5µg; total RNA, 10µg. Summary The RNAgents Total RNA Isolation System provided a rapid and efficient method for isolation of total RNA from different maize tissues. Furthermore, by introducing the described modifications to the standard Promega protocol, this procedure may be applicable to other plant systems containing high concentrations of polysaccharides. We found the PolyATtract System to be a quick method for the isolation of very clean, undegraded poly(a)+ RNA in high yield. The PolyATtract System 1000, in particular, represents a simple and rapid method for the direct isolation of mrnas from maize tissues, and is very convenient when only small amounts of starting material are available for RNA isolation. References Schneiderbauer, A.H. et al. (1992) Anal. Biochem. 197, 9 2. Tesniere, C. and Vayda, M.E. (1991) Plant Mol. Biol. Reptr. 9, Chirgwin, J.M. (1979) Biochem. 18, Logeman, J. et al. (1987) Anal. Biochem. 163, Laroche-Raynal, M. et al (1991) Plant Sci. Lett. 35, RNAgents Total RNA Isolation System Technical Bulletin, #TB087, Promega Corporation. 7. Fang, G.S. et al. (1992) BioTech. 13, Grierson, D. (1982) In: Nucleic Acids and Proteins in Plants, Vol. II, Parthier, B. and Boulter, A., eds., Springer-Verlag, Berlin, PolyATtract mrna Isolation Systems Technical Manual, #TM021, Promega Corporation. 10. Casacuberta, J.M. et al. (1991) Plant Mol. Biol. 16, PolyATtract System 1000 Technical Manual, #TM228, Promega Corporation. Ordering Information

5 Product Cat.# RNAgents Total RNA Isolation System Z5110 PolyATtract mrna Isolation System I Z5210 PolyATtract mrna Isolation System II Z5200 PolyATtract mrna Isolation System III Z5300 PolyATtract mrna Isolation System IV Z5310 PolyATtract System 1000 Z5420 PolyATtract System 1000 Z5400 (without Magnetic Stand) PolyATtract Series 9600(TM) mrna Isolation System Z3790 with cdna Synthesis Reagents PolyATtract Series 9600(TM) mrna Isolation System Z3890 without cdna Synthesis Reagents PolyATtract Series 9600(TM) Multi-Magnet Z Promega Corporation. All Rights Reserved. PolyATract and RNAgents are registered trademarks of Promega Corporation. Series 9600 is a trademark of Promega Corporation Lambda ZAP is a registered trademark of Strategene Cloning System, inc.