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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
ISOLATION OF HIGH-QUALITY RNA FROM GRAINS OF DIFFERENT MAIZE VARIETIES a
Rafael da Silva Messias , Vanessa Galli
a b
a
a
, Julieti Huch Buss ,
c
Joyce Moura Borowski , Leonardo Nora , Sérgio Delmar dos Anjos a
b
e Silva , Rogério Margis & Cesar Valmor Rombaldi a
c
Embrapa Clima Temperado , Pelotas , Rio Grande do Sul , Brasil
b
Universidade Federal do Rio Grande do Sul, Campus do Vale , Porto Alegre , Rio Grande do Sul , Brasil c
Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário , Pelotas , Rio Grande do Sul , Brasil Accepted author version posted online: 08 Jan 2014.Published online: 06 Jun 2014.
To cite this article: Rafael da Silva Messias , Vanessa Galli , Julieti Huch Buss , Joyce Moura Borowski , Leonardo Nora , Sérgio Delmar dos Anjos e Silva , Rogério Margis & Cesar Valmor Rombaldi (2014) ISOLATION OF HIGH-QUALITY RNA FROM GRAINS OF DIFFERENT MAIZE VARIETIES, Preparative Biochemistry and Biotechnology, 44:7, 697-707, DOI: 10.1080/10826068.2013.868355 To link to this article: http://dx.doi.org/10.1080/10826068.2013.868355
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Preparative Biochemistry & Biotechnology, 44:697–707, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.868355
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ISOLATION OF HIGH-QUALITY RNA FROM GRAINS OF DIFFERENT MAIZE VARIETIES
Rafael da Silva Messias,1 Vanessa Galli,1,2 Julieti Huch Buss,1 Joyce Moura Borowski,1 Leonardo Nora,3 Se´rgio Delmar dos Anjos e Silva,1 Roge´rio Margis,2 and Cesar Valmor Rombaldi3 1 Embrapa Clima Temperado, Pelotas, Rio Grande do Sul, Brasil 2 Universidade Federal do Rio Grande do Sul, Campus do Vale, Porto Alegre, Rio Grande do Sul, Brasil 3 Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universita´rio, Pelotas, Rio Grande do Sul, Brasil
& The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and=or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of b-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy. Keywords carotenoid biosynthesis, gene expression, RNA extraction, Zea mays L.
Rafael da Silva Messias and Vanessa Galli contributed equally to this work. Address correspondence to Rafael da Silva Messias, Laborato´rio de Biologia Molecular, Empresa Brasileira de Pesquisa Agropecua´ria Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970, Pelotas–RS, Brasil. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/lpbb.
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INTRODUCTION Maize is an important cereal crop worldwide. The germplasm of maize is characterized by great genetic variability composed by DNA from adapted populations, introduced exotic materials, and landraces (local varieties). Because of this, maize is considered a potential target of biofortification efforts aiming to increase the content of vitamin A precursors.[1,2] These carotenoids are cleaved in the intestinal lumen to produce retinol (vitamin A), an essential micronutrient for human health. The World Health Organization (WHO) estimates that there are more than 250 million children worldwide with disabilities related to vitamin A deficiency, and most cases are found in developing countries.[3] Considering that many of these carotenoids are synthesized directly in the grain, it is essential to characterize the expression of genes associated with carotenoids accumulation in the grain, in order to improve pro-vitamin A through metabolic engineering.[4] However, to perform gene expression studies, it is crucial to isolate high-quality RNA, since the use of low-quality RNA may compromise the reliability of the results of these studies, which are expensive, laborious, and high in cost. High-quality RNA should be intact and free of contamination with RNAses, proteins, genomic DNA, enzymes, and other inhibitory substances for real-time polymerase chain reaction (RT-PCR).[5,6] However, the isolation of RNA from maize grains is notoriously difficult due to its association with the amylo-protein matrix of the endosperm. Thus, co-purification and co-precipitation of polysaccharides, proteins, and polyphenols present in high concentrations in those tissues normally compromise the isolation of RNA regarding its yield, purity, and integrity.[7–9] These contaminants may inhibit reverse transcriptase activity and consequently affect DNA synthesis from isolated RNA. Therefore, the use of an appropriate RNA isolation method is necessary to ensure reliable results.[5,10] Several RNA isolation methods have been evaluated for polysaccharide-rich samples.[8,9,11–13] However, most of them resulted in unsatisfactory quantity and quality of isolated RNA from maize grains. Furthermore, samples from different maize varieties show differences in their composition that could compromise the isolation of RNA using a single protocol. Since the use of different RNA isolation methods to evaluate gene expression makes the procedures more laborious and may result in ambiguous results, the development of a single protocol able to isolate high-quality RNA from different varieties is necessary. As far as we know, the efficiency of different protocols for RNA isolation of maize grains from different varieties has not been evaluated. In this context, a cetyltrimethylammonium bromide (CTAB)-based method adapted from Jakkola et al.[14] and Chang et al.[15] was compared
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with different other methods of RNA isolation, including commercial reagents and RNA kits and DNA kits, in order to find a simple, efficient, and low-cost strategy able to isolate high-quality RNA from maize grains from different varieties. EXPERIMENTAL
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Plant Material Cobs from maize varieties (Zea mays L.) were collected 24 days after pollination. Cobs were immediately frozen in liquid nitrogen in plastic bags previously treated with RNase inhibitor (RNase AWAY, Invitrogen) and kept stored at 70 C until analysis. Immediately before the RNA isolation, the cobs were shelled in a frozen state, and whole grains were grinded in a pestle and mortar using liquid nitrogen. For each method of RNA isolation, four aliquots of 100 mg were prepared in a 1.5-mL microtube (Eppendorf), previously cooled. RNA Isolation Methods The methods evaluated in this study were: M1. CTAB (hexadecyltrimethylammonium bromide), adapted in this study. M2. PureLink Plant RNA Reagent (Invitrogen). M3. TRIzol Reagent (Invitrogen). M4. Nucleo Spin RNA Plant (Macherey-Nagel). M5. Spectrum Plant Total RNA Kit (Sigma Aldrich). M6. RNeasy Plant Mini Kit (Qiagen). M7. PureLink Plant Total DNA Purification Kit (Invitrogen). M1 consists of a CTAB-based method modified from the methods proposed by Jaakola et al.[14] for the isolation of total RNA from bilberry (Vaccinium myrtillus) and Chang et al.[15] for pine tree samples. To perform the CTAB-modified method, the premilled samples (100 mg) were suspended in 1.25 mL of pre-heated (65 C) extraction solution (2% CTAB, 2% polyvinylpyrrolidone [PVP], 2 M NaCl, 100 mM Tris-HCl pH 8.0, 25 mM ethylenediamine tetraacetic acid [EDTA], and 3.3% b-mercaptoethanol) and incubated at 65 C for 3 min. Two extractions were performed with an equal volume of chloroform–isoamyl alcohol (24:1), after centrifugation at 7300 g for 20 min. The upper phase was recovered and precipitated for 1 hr at 70 C with 1=4 of 10 M LiCl. After this period the samples were centrifuged at 18,000 g for 30 min at 4 C. The pellet was resuspended at 65 C
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for a few minutes in 200 mL of SSTE buffer (1 M NaCl, 0.5% sodium dodecyl sulfate [SDS], 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) and extracted twice with an equal volume of chloroform:isoamyl alcohol (24:1). The upper phase was precipitated with 364 mL of 100% ethanol and 18 mL of 3 M sodium acetate, pH 5.5, for 1 hr at 70 C followed by centrifugation at 18,000 g for 20 min at 4 C. The pellet was washed twice with 70% ethanol and resuspended in diethylpyrocarbonate (DEPC)-treated water with 0.1 mM EDTA . The M2 and M3 protocols used commercial reagents. In M2, 1.2 M NaCl and 0.8 M sodium citrate were included in the precipitation step to reduce polysaccharide contamination. Both methods were performed according to the manufacturer’s instructions. M4, M5, and M6 used commercial kits for RNA isolation of plant tissues and were performed according to the manufacturer’s instructions. Due to the high viscosity, RAP lysis buffer was used in M4, and RLC buffer was used in M6; both utilized guanidine hydrochloride, as recommended by the manufacturer’s protocol to reduce solidification of the sample and allow the lysate to flow. M7 is a DNA isolation kit that was used for RNA isolation by replacing the RNAse digestion step with a DNAse digestion step. For all methods the isolated RNA was eluted in 30 mL of DEPC-treated water. To eliminate DNA contamination, 1 mg of total RNA extracted from each method was digested with 1 U DNAse (Invitrogen). Characterization of Isolated RNA The purity and quantity of RNA were determined by monitoring both A260=A280 and A260=A230 absorbance ratios using Nanovue Plus (GE Healthcare). RNA integrity was evaluated from the 25S and 18S ribosomal RNA (rRNA) bands on 1.0% formaldehyde–agarose gel after electrophoresis, stained with gel red, and visualized with ultraviolet (UV) light. The integrity of total RNA isolated with the M1 protocol was also determined by assaying 1 mL of diluted total RNA using Agilent’s 2100 Bioanalyzer with the Plant RNA Picochip assay in accordance with the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA). First-Strand cDNA Synthesis and Real-Time PCR Analysis The first strand cDNA was obtained using 100 ng of total RNA, 1 mL 10 mM dNTP, 1 mL oligo (dT)12-18 (500 mg=mL), 4 mL 5 First-Strand Buffer, 2 mL 0.1 M DTT, 1 mL RNaseOUT (40 units=mL), and 1 mL (200 U) M-MLV enzyme, and incubated according to the manufacturer’s instructions (Invitrogen). The cDNAs were amplified by real-time qualitative
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polymerase chain reaction (RT-qPCR) in a final volume of 20 mL containing 1 mL cDNA, 10 mL of Platinum Sybr green UDG (Invitrogen), and 3 pmol of each primer. The primers for the amplification of tubulin (TUB) gene (F: 50 -AGAACTGCGACTGCCTCCAAAGG-30 , R: 50 -AGATGAGCAGGGTGCCCATTC-30 ) and b carotene hydroxylase (HYD3) gene (F: 50 -GGGGATTA CGCTGTTCGG-30 , R: 50 -GTGGTGTATCTTGTGCGAGG-30 ) were designed using the Vector NTI 11 software based on maize sequences (GenBank J01238.1). Amplification was standardized in a 7500 Real Time Fast Thermal Cycler (Applied Biosystems) using the following conditions: 50 C for 20 sec, 95 C for 10 min, followed by 45 cycles of 15 sec at 95 C and 60 sec at 60 C. No-template controls and a reverse-transcription negative control were included to evaluate the presence of genomic DNA contamination. Relative expression data was calculated according to the 2-DDCt method and presented as fold change.[16]
RESULTS AND DISCUSSION Total RNA from maize grains is usually isolated with commercially available kits, which are costly and result in a gel-like mixture in the extraction solution as well as a water-insoluble pellet, which prevents RNA isolation in several maize varieties that contain high levels of polysaccharides. The quantity and quality of the RNA isolated are important for gene expression analysis; therefore, we modified a CTAB-based protocol and compared this method with six RNA extraction methods to obtain an efficient and inexpensive method. PureLink Plant RNA (M2) is a commercial reagent recommended for RNA isolation from different plant tissues, especially tissues with high levels of polyphenols and sugars. This reagent obtained high amounts of RNA (46.93 11.51 mg=100 mg) from maize grains; however, the A260=A230 ratio showed values below 1.8 (Table 1), suggesting polyphenol and polysaccharide contamination. In addition, the A260=A280 ratio varied largely according to the maize variety, indicating protein contamination in the V3 variety. Similarly, the isolation of RNA with TRIzol Reagent (M3) proved to be difficult due to the formation of a viscous mixture in the extraction buffer and a water-insoluble pellet. These problems seriously affected the purity of the isolated RNA (A260=A230 and A260=A280 ratios below 1.8) and resulted in high variability in the quantity of RNA isolated among sample replicates (Table 1). For example, the coefficient variation in the V3 variety reached 73% using this method, leading to replicate deviation, shown in Figure 1. Therefore, the RNA isolated from maize grains using the M2 and M3 methods was not considered suitable for gene expression analysis, corroborating the results observed in previous studies using polysaccharide-rich samples.[8,9]
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V2
21.59 5.02 38.50 6.00 41.93 30.56 1.12 0.67 2.02 0.19 ND 13.07 4.09
V1
13.82 3.06 46.93 11.51 30.64 7.19 1.62 0.282 6.04 1.36 0.383 0.34 11.66 4.93
V3 33.12 8.15 26.26 3.33 29.10 15.49 1.12 0.22 9.58 1.55 0.46 0.20 34.11 1.93
RNA Yield (mg=100 mg)
2.08 0.03 2.08 0.03 1.62 0.07 1.48 0.04 1.88 0.03 1.41 0.53 2.04 0.06
V1 2.08 0.01 2.05 0.03 1.56 0.12 1.55 0.02 1.95 0.30 ND 2.05 0.06
V2 2.08 0.03 1.19 0.08 1.70 0.07 2.45 0.11 2.07 0.01 2.72 1.79 2.08 0.01
V3
Quality of RNA (A260=A280 Ratio)
2.09 0.02 1.67 0.09 0.31 0.12 0.06 0.03 2.83 1.26 1.98 2.37 2.63 0.33
V1
2.10 0.01 1.61 0.05 0.33 0.05 0.14 0.19 4.52 2.25 ND 2.59 0.32
V2
2.18 0.04 1.20 0.04 0.37 0.06 0.06 0.03 2.65 0.07 0.10 0.10 2.34 0.01
V3
Quality of RNA (A260=A230 Ratio)
Yield and Quality of Total RNA Isolated From Grains of Three Maize Varieties Using Seven Isolation Methods
Note. V1, V2, and V3: maize varieties. ND, not detected. Mean standard deviation of four replicates.
M1 M2 M3 M4 M5 M6 M7
Method
TABLE 1
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FIGURE 1 Seven methods to isolate RNA from grains of V3 variety. (a) Electrophoresis in 1.0% formaldehyde–agarose gel showing RNA isolated with the seven methods (n ¼ 3). (b) Electrophoresis in 3.0% agarose gel showing amplification of TUB gene using cDNA (lanes 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, and 20) and reverse transcription negative control (lanes 3, 6, 9, 12, 15, 18, and 21) obtained with the seven methods. (c) Expression of HYD3 by RT-qPCR, using RNA from methods M1, M2, M3, M5, and M7. M1, CTAB adapted in the present study; M2. PureLink Plant RNA Reagent (Invitrogen); M3, TRIzol Reagent (Invitrogen); M4, Nucleo Spin RNA Plant (Macherey-Nagel); M5, Spectrum Plant Total RNA Kit (Sigma Aldrich); M6, RNeasy Plant Mini Kit (Qiagen); M7, PureLink Plant Total DNA Purification Kit (Invitrogen).
RNA isolation kits combine the selective binding properties of a silica gel membrane and the use of a rapid spin. The quantity and quality of RNA isolated from maize grains with the three RNA isolation kits (M4, M5, and M6) were low compared to the other methods (Figure 1a and Table 1), most likely because of the high viscosity of the generated extract, which prevented its flow through the columns provided in these kits. Therefore, these kits were not considered appropriate to isolate RNA from maize grains. Another evaluated approach was the use of commercial kits for DNA isolation, which are quite numerous. DNA isolation kits are more practical and cheaper than RNA isolation kits. In the present study, M7 efficiently isolated high-quality RNA in an amount sufficient for use in RT-qPCR analysis (Figure 1a and Table 1). The efficiency of this strategy was previously described by Belefant-Miller et al.[12] using rice samples. Similar to the DNA isolation kit, the CTAB method modified in the present study (M1) yielded satisfactory results. This method used CTAB as the detergent buffer to separate polysaccharides from the nucleic acids, and PVP and b-mercaptoethanol were used to reduce phenolic compound oxidation. This method also separated the sugars present in the samples through LiCl precipitation followed by centrifugation. To obtain high-quality RNA from maize grains, several modifications from the methods proposed by Jaakola et al.[14] and Chang et al.[15] were conducted. The modifications included a reduction in the weight of the sample and, consequently, a reduction in the extraction volume, allowing all the steps to be performed in microcentrifuge tubes, which reduced the length of time necessary to complete the protocol without decreasing the RNA yield.
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Furthermore, the use of spermidine in the extraction buffer and the extraction step with phenol:chloroform:isoamyl alcohol after the addition of SSTE were suppressed from the original protocol. Additionally, the samples were incubated with LiCl for 1 hr at 70 C instead of overnight at 4 C, and the RNA was further precipitated with sodium acetate and ethanol instead of only ethanol, making the RNA less hydrophilic, resulting in efficient RNA precipitation by increasing the salt concentration. Finally, the RNA was washed with ethanol to remove residual contaminants. Moreover, the number of centrifugation and washing steps was reduced, which resulted in a less laborious method. These modifications effectively maintained the carbohydrates in a soluble form and resulted in a RNA pellet that was easily redissolved in water. As a result, this method provided highly pure total RNA relatively free of contamination with proteins, carbohydrates, and phenolic compounds, as observed by the A260=A230 and A260=A280 ratios, as well as RNA concentrations desirable for gene expression studies (Table 1). Intact bands corresponding to 25S and 18S rRNA were also observed on a formaldehyde agarose gel, indicating that little or no RNA degradation occurred during extraction compared to the other methods evaluated (Figure 1a). The integrity of RNA molecules is of paramount importance for the subsequent gene expression experiments; therefore, we also assessed the integrity of total RNA isolated from six random maize varieties with the CTAB method using an Agilent 2100 Bioanalyzer (Figure 2 and Supplementary Figure 1). The bioanalyzer separates RNA fragments by size using an electrophoretic technology on a chip; the RNA fragments are
FIGURE 2 (A) Electrophoresis of total RNA isolated with the CTAB method (M1) from maize grains of six varieties on the Agilent 2100 Bioanalyzer using the Plant RNA Pico Assay. (B) Electropherogram of sample 3, provided as an example.
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read by laser-induced fluorescence and translated into gel-like bands and peaks (electropherograms). Therefore, it is possible to measure the RNA integrity using the ratio of the large (25S) to small (18S) ribosomal RNA subunits (25S=18S) and the RNA integrity number (RIN), which estimates the integrity of the entire RNA profile, including 25S=18S, and the presence and absence of other electropherogram peaks through a regression model.[17] The total RNA isolated with the CTAB method showed RIN values ranging from 6.9 to 8.5, and the 25S=18S ratios were between 1.8 and 3.2 (Supplementary Figure 1). The electropherograms and gel-like images from one of the RNA samples are exemplified in Figure 2, showing that the total RNA isolated from maize varieties with the CTAB method is of high quality. This protocol was also tested with 25 other varieties, which resulted in pure RNA, with A260=A280 ¼ 1.91 0.12 and A260= A230 ¼ 2.12 0.15 (mean standard deviation), in an amount sufficient to be analyzed in RT-qPCR studies (32.05 7.01 mg=100 mg); thus, this is a highly reproducible method to use with grains with different polysaccharide levels. Therefore, the quantity and quality of total RNA extracted with the CTAB method were similar to those from M7 and represented a reduction in the experimental costs. Other methods using CTAB in the extraction buffer have previously been used for the extraction of RNA from polysaccharide-enriched samples.[18–20] However, only one recently published protocol isolated RNA from maize grains and obtained satisfactory results similar to those in the present study.[21] However, this study did not evaluate the extraction efficiency using grains from different varieties, and the protocol evaluated was based on the addition of buffers and several steps before and after TRIzol extraction and therefore still depended on the use of commercial reagents, which increased the experimental costs. Finally, the expression of HYD3 (associated with carotenoid biosynthesis) was assessed using the same amount of RNA from all methods to evaluate the impact of using the appropriate RNA isolation method in the RT-qPCR assays. As observed in Figure 1c, the transcript level was different between the evaluated methods, confirming the importance of using the same RNA isolation method to compare gene expression. Furthermore, a high deviation of transcript levels between replicates was observed using the RNA isolated using the M2, M3, and M5 methods, most likely because of the low purity of the isolated RNA. The TUB transcripts in the RNA from M6 showed a high variation among replicates after cycle 36, most likely because of the low quality of isolated RNA, which impaired the quantification of the HYD3 transcripts. The presence of residual DNA contamination in the RNA isolated with these methods was also evaluated. To this end, the TUB primers were designed in two different exons, resulting in amplicons of different sizes when cDNA or DNA is amplified. Therefore, using samples previously
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digested with DNAse but not transcribed to cDNA in the RT-qPCR, a second melting peak was observed that corresponded to a band with a higher molecular weight in the agarose gel after electrophoresis in samples from M3 and M5, indicating the presence of residual DNA (Figure 2b), which results in the overestimation of gene expression. In contrast, the use of high-quality RNA isolated from M1 and M7 resulted in similar expression patterns, with reproducible and specific amplification products and with minor variation among extraction replicates (Figure 1c), confirming the importance of using the appropriate RNA isolation method to obtain reliable results. CONCLUSIONS The results of this study indicate that the method of isolation directly affects the quality and quantity of RNA obtained from maize grains. The CTAB method overcomes the problems associated with different levels of polysaccharides in maize varieties, and is a practical and economically feasible method that can be routinely performed in any laboratory. The RNA isolated with this method showed high quality (purity and integrity) and yield, which were reproducible, and resulted in efficient and reliable results in RT-qPCR analysis, as confirmed by the amplification of HYD3 gene. SUPPLEMENTAL MATERIAL Supplemental data for this article can be accessed on the publisher’s website. REFERENCES 1. Aluru, M.; Xu, Y.; Guo, R.; Wang, Z.; Li, S.; White, W.; Wang, C.; Rodermel, S. Generation of Transgenic Maize With Enhanced Provitamin A Content. J. Exp. Bot. 2008, 59, 3551–3562. 2. Berardo, N.; Mazzinelli, G.; Valoti, P.; Lagana, P.; Redaellij, R. Characterization of Maize Germplasm for the Chemical Composition of the Grain. Agriculture and Food Chemistry. 2009, 57, 2378–2384. 3. World Health Organization. Global Prevalence of Vitamin A Deficiency in Populations at Risk 1995–2005. In WHO Global Database on Vitamin A Deficiency. World Health Organization: Geneva, Switzerland, 2009. Available at: http://www.who.int/vmnis/database/vitamina/x/en/. 4. Vallabhaneni, R.; Wurtzel, E.T. Timing and Biosynthetic Potential for Carotenoid Accumulation in Genetically Diverse Germplasm of Maize. Plant Physiol. 2009, 150, 562–572. 5. Bustin, S.A.; Nolan, T. Pitfalls of Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction. J. Biomol. Techniques 2004, 15, 155–166. 6. Fleige, S.; Pfaffl, M.W. RNA Integrity and the Effect on the Real-Time qRT-PCR Performance. Mol. Aspects Med. 2006, 27, 126–139. 7. Rossen, L.; Norskov, P.; Holmstrom, K.; Rasmussen, O.F. Inhibition of PCR by Components of Food Samples, Microbial Diagnostic Assays and DNA-Extraction Solutions. Int. J. Food Microbiol. 1992, 17, 37–45.
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8. Coan ˜ a, Y.P.; Parody, N.; Ferna´ndez-Caldas, E.; Alonso, C. A Modified Protocol for RNA Isolation From High Polysaccharide Containing Cupressus arizonica Pollen. Applications for RT–PCR and Phage Display Library Construction. Mol. Biotechnol. 2010, 44, 127–132. 9. Rubio-Pin ˜ a, J.A.; Zapata-Pe´rez, O. Isolation of Total RNA From Tissues rich in Polyphenols and Polysaccharides of Mangrove Plants. Electron. J. Biotechnol. 2011, 14. 10. Wang, E. RNA Amplification for Successful Gene Profiling Analysis. J. Translational Med. 2005, 3. 11. Wang, D.H.; Wang, B.C.; Li, B.; Duan, C.R.; Zhang, J. Extraction of Total RNA From Chrysanthemum Containing High Levels of Phenolic and Carbohydrates. Colloid Surf. Biointerfaces 2004, 36, 111–114. 12. Belefant-Miller, H.; Ledbetter, C.; Bennett, S. Using a Commercial DNA Extraction Kit to Obtain RNA for RT-PCR From Starchy Rice Endosperm. Biotechnol. J. 2008, 3, 360–363. 13. Zhang, Y.; Qin, Y.; Guo, L.; Zhou, Z.; Liang, Z.; Zhang, C.; Guo, H. Isolation of High Quality RNA From Polyporus umbellatus (Pers.) Fries. Electron. J. Biotechnol. 2012, 15. 14. Jaakola, L.; Pirttila, A.M.; Halonen, M.; Hohtola, A. Isolation of High Quality RNA From Bilberry (Vaccinium myrtillus L.). Fruit. Mol. Biotechnol. 2001, 19, 201–203. 15. Chang, S.; Puryear, J.; Cairney, J. A Simple and Efficient Method for Isolating RNA From Pine Trees. Plant Mol. Biol. Rep. 1993, 11, 113–116. 16. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-DeltaDeltaCT Method. Methods 2001, 25, 402–408. 17. Schroeder, A.; Mueller, O.; Stocker, S.; Salowsky, R.; Leiber, M.; et al. The RIN: An RNA Integrity Number for Assigning Integrity Values to RNA Measurements. BMC Mol. Biol. 2006, 7, 3. 18. Zeng, Y.; Yang, T. RNA Isolation From Highly Viscous Samples Rich in Polyphenols and Polysaccharides. Plant Mol. Biol. Rep. 2002, 20, 417. 19. Wang, W.; Stegemann, J.P. Extraction of High Quality RNA From Polysaccharide Matrices Using Cetlytrimethylammonium Bromide. Biomaterials 2010, 31, 1612–1618. 20. Zhao, L.; Ding, Q.; Zeng, J.; Wang, F.R.; Zhang, J.; Fan, S.J.; He, X.Q. An Improved CTAB– Ammonium Acetate Method for Total RNA Isolation From Cotton. Phytochem. Anal. 2012, 23, 647–650. 21. Wang, G.; Wang, G.; Zhang, X.; Wang, F.; Song, R. Isolation of High Quality RNA From Cereal Seeds Containing High Levels of Starch. Phytochem. Anal. 2011, 23, 159–163.
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
ISOLATION OF HIGH-QUALITY RNA FROM GRAINS OF DIFFERENT MAIZE VARIETIES a
Rafael da Silva Messias , Vanessa Galli
a b
a
a
, Julieti Huch Buss ,
c
Joyce Moura Borowski , Leonardo Nora , Sérgio Delmar dos Anjos a
b
e Silva , Rogério Margis & Cesar Valmor Rombaldi a
c
Embrapa Clima Temperado , Pelotas , Rio Grande do Sul , Brasil
b
Universidade Federal do Rio Grande do Sul, Campus do Vale , Porto Alegre , Rio Grande do Sul , Brasil c
Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário , Pelotas , Rio Grande do Sul , Brasil Accepted author version posted online: 08 Jan 2014.Published online: 06 Jun 2014.
To cite this article: Rafael da Silva Messias , Vanessa Galli , Julieti Huch Buss , Joyce Moura Borowski , Leonardo Nora , Sérgio Delmar dos Anjos e Silva , Rogério Margis & Cesar Valmor Rombaldi (2014) ISOLATION OF HIGH-QUALITY RNA FROM GRAINS OF DIFFERENT MAIZE VARIETIES, Preparative Biochemistry and Biotechnology, 44:7, 697-707, DOI: 10.1080/10826068.2013.868355 To link to this article: http://dx.doi.org/10.1080/10826068.2013.868355
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Preparative Biochemistry & Biotechnology, 44:697–707, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.868355
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ISOLATION OF HIGH-QUALITY RNA FROM GRAINS OF DIFFERENT MAIZE VARIETIES
Rafael da Silva Messias,1 Vanessa Galli,1,2 Julieti Huch Buss,1 Joyce Moura Borowski,1 Leonardo Nora,3 Se´rgio Delmar dos Anjos e Silva,1 Roge´rio Margis,2 and Cesar Valmor Rombaldi3 1 Embrapa Clima Temperado, Pelotas, Rio Grande do Sul, Brasil 2 Universidade Federal do Rio Grande do Sul, Campus do Vale, Porto Alegre, Rio Grande do Sul, Brasil 3 Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universita´rio, Pelotas, Rio Grande do Sul, Brasil
& The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and=or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of b-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy. Keywords carotenoid biosynthesis, gene expression, RNA extraction, Zea mays L.
Rafael da Silva Messias and Vanessa Galli contributed equally to this work. Address correspondence to Rafael da Silva Messias, Laborato´rio de Biologia Molecular, Empresa Brasileira de Pesquisa Agropecua´ria Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970, Pelotas–RS, Brasil. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/lpbb.
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INTRODUCTION Maize is an important cereal crop worldwide. The germplasm of maize is characterized by great genetic variability composed by DNA from adapted populations, introduced exotic materials, and landraces (local varieties). Because of this, maize is considered a potential target of biofortification efforts aiming to increase the content of vitamin A precursors.[1,2] These carotenoids are cleaved in the intestinal lumen to produce retinol (vitamin A), an essential micronutrient for human health. The World Health Organization (WHO) estimates that there are more than 250 million children worldwide with disabilities related to vitamin A deficiency, and most cases are found in developing countries.[3] Considering that many of these carotenoids are synthesized directly in the grain, it is essential to characterize the expression of genes associated with carotenoids accumulation in the grain, in order to improve pro-vitamin A through metabolic engineering.[4] However, to perform gene expression studies, it is crucial to isolate high-quality RNA, since the use of low-quality RNA may compromise the reliability of the results of these studies, which are expensive, laborious, and high in cost. High-quality RNA should be intact and free of contamination with RNAses, proteins, genomic DNA, enzymes, and other inhibitory substances for real-time polymerase chain reaction (RT-PCR).[5,6] However, the isolation of RNA from maize grains is notoriously difficult due to its association with the amylo-protein matrix of the endosperm. Thus, co-purification and co-precipitation of polysaccharides, proteins, and polyphenols present in high concentrations in those tissues normally compromise the isolation of RNA regarding its yield, purity, and integrity.[7–9] These contaminants may inhibit reverse transcriptase activity and consequently affect DNA synthesis from isolated RNA. Therefore, the use of an appropriate RNA isolation method is necessary to ensure reliable results.[5,10] Several RNA isolation methods have been evaluated for polysaccharide-rich samples.[8,9,11–13] However, most of them resulted in unsatisfactory quantity and quality of isolated RNA from maize grains. Furthermore, samples from different maize varieties show differences in their composition that could compromise the isolation of RNA using a single protocol. Since the use of different RNA isolation methods to evaluate gene expression makes the procedures more laborious and may result in ambiguous results, the development of a single protocol able to isolate high-quality RNA from different varieties is necessary. As far as we know, the efficiency of different protocols for RNA isolation of maize grains from different varieties has not been evaluated. In this context, a cetyltrimethylammonium bromide (CTAB)-based method adapted from Jakkola et al.[14] and Chang et al.[15] was compared
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with different other methods of RNA isolation, including commercial reagents and RNA kits and DNA kits, in order to find a simple, efficient, and low-cost strategy able to isolate high-quality RNA from maize grains from different varieties. EXPERIMENTAL
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Plant Material Cobs from maize varieties (Zea mays L.) were collected 24 days after pollination. Cobs were immediately frozen in liquid nitrogen in plastic bags previously treated with RNase inhibitor (RNase AWAY, Invitrogen) and kept stored at 70 C until analysis. Immediately before the RNA isolation, the cobs were shelled in a frozen state, and whole grains were grinded in a pestle and mortar using liquid nitrogen. For each method of RNA isolation, four aliquots of 100 mg were prepared in a 1.5-mL microtube (Eppendorf), previously cooled. RNA Isolation Methods The methods evaluated in this study were: M1. CTAB (hexadecyltrimethylammonium bromide), adapted in this study. M2. PureLink Plant RNA Reagent (Invitrogen). M3. TRIzol Reagent (Invitrogen). M4. Nucleo Spin RNA Plant (Macherey-Nagel). M5. Spectrum Plant Total RNA Kit (Sigma Aldrich). M6. RNeasy Plant Mini Kit (Qiagen). M7. PureLink Plant Total DNA Purification Kit (Invitrogen). M1 consists of a CTAB-based method modified from the methods proposed by Jaakola et al.[14] for the isolation of total RNA from bilberry (Vaccinium myrtillus) and Chang et al.[15] for pine tree samples. To perform the CTAB-modified method, the premilled samples (100 mg) were suspended in 1.25 mL of pre-heated (65 C) extraction solution (2% CTAB, 2% polyvinylpyrrolidone [PVP], 2 M NaCl, 100 mM Tris-HCl pH 8.0, 25 mM ethylenediamine tetraacetic acid [EDTA], and 3.3% b-mercaptoethanol) and incubated at 65 C for 3 min. Two extractions were performed with an equal volume of chloroform–isoamyl alcohol (24:1), after centrifugation at 7300 g for 20 min. The upper phase was recovered and precipitated for 1 hr at 70 C with 1=4 of 10 M LiCl. After this period the samples were centrifuged at 18,000 g for 30 min at 4 C. The pellet was resuspended at 65 C
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for a few minutes in 200 mL of SSTE buffer (1 M NaCl, 0.5% sodium dodecyl sulfate [SDS], 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) and extracted twice with an equal volume of chloroform:isoamyl alcohol (24:1). The upper phase was precipitated with 364 mL of 100% ethanol and 18 mL of 3 M sodium acetate, pH 5.5, for 1 hr at 70 C followed by centrifugation at 18,000 g for 20 min at 4 C. The pellet was washed twice with 70% ethanol and resuspended in diethylpyrocarbonate (DEPC)-treated water with 0.1 mM EDTA . The M2 and M3 protocols used commercial reagents. In M2, 1.2 M NaCl and 0.8 M sodium citrate were included in the precipitation step to reduce polysaccharide contamination. Both methods were performed according to the manufacturer’s instructions. M4, M5, and M6 used commercial kits for RNA isolation of plant tissues and were performed according to the manufacturer’s instructions. Due to the high viscosity, RAP lysis buffer was used in M4, and RLC buffer was used in M6; both utilized guanidine hydrochloride, as recommended by the manufacturer’s protocol to reduce solidification of the sample and allow the lysate to flow. M7 is a DNA isolation kit that was used for RNA isolation by replacing the RNAse digestion step with a DNAse digestion step. For all methods the isolated RNA was eluted in 30 mL of DEPC-treated water. To eliminate DNA contamination, 1 mg of total RNA extracted from each method was digested with 1 U DNAse (Invitrogen). Characterization of Isolated RNA The purity and quantity of RNA were determined by monitoring both A260=A280 and A260=A230 absorbance ratios using Nanovue Plus (GE Healthcare). RNA integrity was evaluated from the 25S and 18S ribosomal RNA (rRNA) bands on 1.0% formaldehyde–agarose gel after electrophoresis, stained with gel red, and visualized with ultraviolet (UV) light. The integrity of total RNA isolated with the M1 protocol was also determined by assaying 1 mL of diluted total RNA using Agilent’s 2100 Bioanalyzer with the Plant RNA Picochip assay in accordance with the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA). First-Strand cDNA Synthesis and Real-Time PCR Analysis The first strand cDNA was obtained using 100 ng of total RNA, 1 mL 10 mM dNTP, 1 mL oligo (dT)12-18 (500 mg=mL), 4 mL 5 First-Strand Buffer, 2 mL 0.1 M DTT, 1 mL RNaseOUT (40 units=mL), and 1 mL (200 U) M-MLV enzyme, and incubated according to the manufacturer’s instructions (Invitrogen). The cDNAs were amplified by real-time qualitative
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polymerase chain reaction (RT-qPCR) in a final volume of 20 mL containing 1 mL cDNA, 10 mL of Platinum Sybr green UDG (Invitrogen), and 3 pmol of each primer. The primers for the amplification of tubulin (TUB) gene (F: 50 -AGAACTGCGACTGCCTCCAAAGG-30 , R: 50 -AGATGAGCAGGGTGCCCATTC-30 ) and b carotene hydroxylase (HYD3) gene (F: 50 -GGGGATTA CGCTGTTCGG-30 , R: 50 -GTGGTGTATCTTGTGCGAGG-30 ) were designed using the Vector NTI 11 software based on maize sequences (GenBank J01238.1). Amplification was standardized in a 7500 Real Time Fast Thermal Cycler (Applied Biosystems) using the following conditions: 50 C for 20 sec, 95 C for 10 min, followed by 45 cycles of 15 sec at 95 C and 60 sec at 60 C. No-template controls and a reverse-transcription negative control were included to evaluate the presence of genomic DNA contamination. Relative expression data was calculated according to the 2-DDCt method and presented as fold change.[16]
RESULTS AND DISCUSSION Total RNA from maize grains is usually isolated with commercially available kits, which are costly and result in a gel-like mixture in the extraction solution as well as a water-insoluble pellet, which prevents RNA isolation in several maize varieties that contain high levels of polysaccharides. The quantity and quality of the RNA isolated are important for gene expression analysis; therefore, we modified a CTAB-based protocol and compared this method with six RNA extraction methods to obtain an efficient and inexpensive method. PureLink Plant RNA (M2) is a commercial reagent recommended for RNA isolation from different plant tissues, especially tissues with high levels of polyphenols and sugars. This reagent obtained high amounts of RNA (46.93 11.51 mg=100 mg) from maize grains; however, the A260=A230 ratio showed values below 1.8 (Table 1), suggesting polyphenol and polysaccharide contamination. In addition, the A260=A280 ratio varied largely according to the maize variety, indicating protein contamination in the V3 variety. Similarly, the isolation of RNA with TRIzol Reagent (M3) proved to be difficult due to the formation of a viscous mixture in the extraction buffer and a water-insoluble pellet. These problems seriously affected the purity of the isolated RNA (A260=A230 and A260=A280 ratios below 1.8) and resulted in high variability in the quantity of RNA isolated among sample replicates (Table 1). For example, the coefficient variation in the V3 variety reached 73% using this method, leading to replicate deviation, shown in Figure 1. Therefore, the RNA isolated from maize grains using the M2 and M3 methods was not considered suitable for gene expression analysis, corroborating the results observed in previous studies using polysaccharide-rich samples.[8,9]
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V2
21.59 5.02 38.50 6.00 41.93 30.56 1.12 0.67 2.02 0.19 ND 13.07 4.09
V1
13.82 3.06 46.93 11.51 30.64 7.19 1.62 0.282 6.04 1.36 0.383 0.34 11.66 4.93
V3 33.12 8.15 26.26 3.33 29.10 15.49 1.12 0.22 9.58 1.55 0.46 0.20 34.11 1.93
RNA Yield (mg=100 mg)
2.08 0.03 2.08 0.03 1.62 0.07 1.48 0.04 1.88 0.03 1.41 0.53 2.04 0.06
V1 2.08 0.01 2.05 0.03 1.56 0.12 1.55 0.02 1.95 0.30 ND 2.05 0.06
V2 2.08 0.03 1.19 0.08 1.70 0.07 2.45 0.11 2.07 0.01 2.72 1.79 2.08 0.01
V3
Quality of RNA (A260=A280 Ratio)
2.09 0.02 1.67 0.09 0.31 0.12 0.06 0.03 2.83 1.26 1.98 2.37 2.63 0.33
V1
2.10 0.01 1.61 0.05 0.33 0.05 0.14 0.19 4.52 2.25 ND 2.59 0.32
V2
2.18 0.04 1.20 0.04 0.37 0.06 0.06 0.03 2.65 0.07 0.10 0.10 2.34 0.01
V3
Quality of RNA (A260=A230 Ratio)
Yield and Quality of Total RNA Isolated From Grains of Three Maize Varieties Using Seven Isolation Methods
Note. V1, V2, and V3: maize varieties. ND, not detected. Mean standard deviation of four replicates.
M1 M2 M3 M4 M5 M6 M7
Method
TABLE 1
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FIGURE 1 Seven methods to isolate RNA from grains of V3 variety. (a) Electrophoresis in 1.0% formaldehyde–agarose gel showing RNA isolated with the seven methods (n ¼ 3). (b) Electrophoresis in 3.0% agarose gel showing amplification of TUB gene using cDNA (lanes 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, and 20) and reverse transcription negative control (lanes 3, 6, 9, 12, 15, 18, and 21) obtained with the seven methods. (c) Expression of HYD3 by RT-qPCR, using RNA from methods M1, M2, M3, M5, and M7. M1, CTAB adapted in the present study; M2. PureLink Plant RNA Reagent (Invitrogen); M3, TRIzol Reagent (Invitrogen); M4, Nucleo Spin RNA Plant (Macherey-Nagel); M5, Spectrum Plant Total RNA Kit (Sigma Aldrich); M6, RNeasy Plant Mini Kit (Qiagen); M7, PureLink Plant Total DNA Purification Kit (Invitrogen).
RNA isolation kits combine the selective binding properties of a silica gel membrane and the use of a rapid spin. The quantity and quality of RNA isolated from maize grains with the three RNA isolation kits (M4, M5, and M6) were low compared to the other methods (Figure 1a and Table 1), most likely because of the high viscosity of the generated extract, which prevented its flow through the columns provided in these kits. Therefore, these kits were not considered appropriate to isolate RNA from maize grains. Another evaluated approach was the use of commercial kits for DNA isolation, which are quite numerous. DNA isolation kits are more practical and cheaper than RNA isolation kits. In the present study, M7 efficiently isolated high-quality RNA in an amount sufficient for use in RT-qPCR analysis (Figure 1a and Table 1). The efficiency of this strategy was previously described by Belefant-Miller et al.[12] using rice samples. Similar to the DNA isolation kit, the CTAB method modified in the present study (M1) yielded satisfactory results. This method used CTAB as the detergent buffer to separate polysaccharides from the nucleic acids, and PVP and b-mercaptoethanol were used to reduce phenolic compound oxidation. This method also separated the sugars present in the samples through LiCl precipitation followed by centrifugation. To obtain high-quality RNA from maize grains, several modifications from the methods proposed by Jaakola et al.[14] and Chang et al.[15] were conducted. The modifications included a reduction in the weight of the sample and, consequently, a reduction in the extraction volume, allowing all the steps to be performed in microcentrifuge tubes, which reduced the length of time necessary to complete the protocol without decreasing the RNA yield.
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Furthermore, the use of spermidine in the extraction buffer and the extraction step with phenol:chloroform:isoamyl alcohol after the addition of SSTE were suppressed from the original protocol. Additionally, the samples were incubated with LiCl for 1 hr at 70 C instead of overnight at 4 C, and the RNA was further precipitated with sodium acetate and ethanol instead of only ethanol, making the RNA less hydrophilic, resulting in efficient RNA precipitation by increasing the salt concentration. Finally, the RNA was washed with ethanol to remove residual contaminants. Moreover, the number of centrifugation and washing steps was reduced, which resulted in a less laborious method. These modifications effectively maintained the carbohydrates in a soluble form and resulted in a RNA pellet that was easily redissolved in water. As a result, this method provided highly pure total RNA relatively free of contamination with proteins, carbohydrates, and phenolic compounds, as observed by the A260=A230 and A260=A280 ratios, as well as RNA concentrations desirable for gene expression studies (Table 1). Intact bands corresponding to 25S and 18S rRNA were also observed on a formaldehyde agarose gel, indicating that little or no RNA degradation occurred during extraction compared to the other methods evaluated (Figure 1a). The integrity of RNA molecules is of paramount importance for the subsequent gene expression experiments; therefore, we also assessed the integrity of total RNA isolated from six random maize varieties with the CTAB method using an Agilent 2100 Bioanalyzer (Figure 2 and Supplementary Figure 1). The bioanalyzer separates RNA fragments by size using an electrophoretic technology on a chip; the RNA fragments are
FIGURE 2 (A) Electrophoresis of total RNA isolated with the CTAB method (M1) from maize grains of six varieties on the Agilent 2100 Bioanalyzer using the Plant RNA Pico Assay. (B) Electropherogram of sample 3, provided as an example.
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read by laser-induced fluorescence and translated into gel-like bands and peaks (electropherograms). Therefore, it is possible to measure the RNA integrity using the ratio of the large (25S) to small (18S) ribosomal RNA subunits (25S=18S) and the RNA integrity number (RIN), which estimates the integrity of the entire RNA profile, including 25S=18S, and the presence and absence of other electropherogram peaks through a regression model.[17] The total RNA isolated with the CTAB method showed RIN values ranging from 6.9 to 8.5, and the 25S=18S ratios were between 1.8 and 3.2 (Supplementary Figure 1). The electropherograms and gel-like images from one of the RNA samples are exemplified in Figure 2, showing that the total RNA isolated from maize varieties with the CTAB method is of high quality. This protocol was also tested with 25 other varieties, which resulted in pure RNA, with A260=A280 ¼ 1.91 0.12 and A260= A230 ¼ 2.12 0.15 (mean standard deviation), in an amount sufficient to be analyzed in RT-qPCR studies (32.05 7.01 mg=100 mg); thus, this is a highly reproducible method to use with grains with different polysaccharide levels. Therefore, the quantity and quality of total RNA extracted with the CTAB method were similar to those from M7 and represented a reduction in the experimental costs. Other methods using CTAB in the extraction buffer have previously been used for the extraction of RNA from polysaccharide-enriched samples.[18–20] However, only one recently published protocol isolated RNA from maize grains and obtained satisfactory results similar to those in the present study.[21] However, this study did not evaluate the extraction efficiency using grains from different varieties, and the protocol evaluated was based on the addition of buffers and several steps before and after TRIzol extraction and therefore still depended on the use of commercial reagents, which increased the experimental costs. Finally, the expression of HYD3 (associated with carotenoid biosynthesis) was assessed using the same amount of RNA from all methods to evaluate the impact of using the appropriate RNA isolation method in the RT-qPCR assays. As observed in Figure 1c, the transcript level was different between the evaluated methods, confirming the importance of using the same RNA isolation method to compare gene expression. Furthermore, a high deviation of transcript levels between replicates was observed using the RNA isolated using the M2, M3, and M5 methods, most likely because of the low purity of the isolated RNA. The TUB transcripts in the RNA from M6 showed a high variation among replicates after cycle 36, most likely because of the low quality of isolated RNA, which impaired the quantification of the HYD3 transcripts. The presence of residual DNA contamination in the RNA isolated with these methods was also evaluated. To this end, the TUB primers were designed in two different exons, resulting in amplicons of different sizes when cDNA or DNA is amplified. Therefore, using samples previously
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digested with DNAse but not transcribed to cDNA in the RT-qPCR, a second melting peak was observed that corresponded to a band with a higher molecular weight in the agarose gel after electrophoresis in samples from M3 and M5, indicating the presence of residual DNA (Figure 2b), which results in the overestimation of gene expression. In contrast, the use of high-quality RNA isolated from M1 and M7 resulted in similar expression patterns, with reproducible and specific amplification products and with minor variation among extraction replicates (Figure 1c), confirming the importance of using the appropriate RNA isolation method to obtain reliable results. CONCLUSIONS The results of this study indicate that the method of isolation directly affects the quality and quantity of RNA obtained from maize grains. The CTAB method overcomes the problems associated with different levels of polysaccharides in maize varieties, and is a practical and economically feasible method that can be routinely performed in any laboratory. The RNA isolated with this method showed high quality (purity and integrity) and yield, which were reproducible, and resulted in efficient and reliable results in RT-qPCR analysis, as confirmed by the amplification of HYD3 gene. SUPPLEMENTAL MATERIAL Supplemental data for this article can be accessed on the publisher’s website. REFERENCES 1. Aluru, M.; Xu, Y.; Guo, R.; Wang, Z.; Li, S.; White, W.; Wang, C.; Rodermel, S. Generation of Transgenic Maize With Enhanced Provitamin A Content. J. Exp. Bot. 2008, 59, 3551–3562. 2. Berardo, N.; Mazzinelli, G.; Valoti, P.; Lagana, P.; Redaellij, R. Characterization of Maize Germplasm for the Chemical Composition of the Grain. Agriculture and Food Chemistry. 2009, 57, 2378–2384. 3. World Health Organization. Global Prevalence of Vitamin A Deficiency in Populations at Risk 1995–2005. In WHO Global Database on Vitamin A Deficiency. World Health Organization: Geneva, Switzerland, 2009. Available at: http://www.who.int/vmnis/database/vitamina/x/en/. 4. Vallabhaneni, R.; Wurtzel, E.T. Timing and Biosynthetic Potential for Carotenoid Accumulation in Genetically Diverse Germplasm of Maize. Plant Physiol. 2009, 150, 562–572. 5. Bustin, S.A.; Nolan, T. Pitfalls of Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction. J. Biomol. Techniques 2004, 15, 155–166. 6. Fleige, S.; Pfaffl, M.W. RNA Integrity and the Effect on the Real-Time qRT-PCR Performance. Mol. Aspects Med. 2006, 27, 126–139. 7. Rossen, L.; Norskov, P.; Holmstrom, K.; Rasmussen, O.F. Inhibition of PCR by Components of Food Samples, Microbial Diagnostic Assays and DNA-Extraction Solutions. Int. J. Food Microbiol. 1992, 17, 37–45.
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8. Coan ˜ a, Y.P.; Parody, N.; Ferna´ndez-Caldas, E.; Alonso, C. A Modified Protocol for RNA Isolation From High Polysaccharide Containing Cupressus arizonica Pollen. Applications for RT–PCR and Phage Display Library Construction. Mol. Biotechnol. 2010, 44, 127–132. 9. Rubio-Pin ˜ a, J.A.; Zapata-Pe´rez, O. Isolation of Total RNA From Tissues rich in Polyphenols and Polysaccharides of Mangrove Plants. Electron. J. Biotechnol. 2011, 14. 10. Wang, E. RNA Amplification for Successful Gene Profiling Analysis. J. Translational Med. 2005, 3. 11. Wang, D.H.; Wang, B.C.; Li, B.; Duan, C.R.; Zhang, J. Extraction of Total RNA From Chrysanthemum Containing High Levels of Phenolic and Carbohydrates. Colloid Surf. Biointerfaces 2004, 36, 111–114. 12. Belefant-Miller, H.; Ledbetter, C.; Bennett, S. Using a Commercial DNA Extraction Kit to Obtain RNA for RT-PCR From Starchy Rice Endosperm. Biotechnol. J. 2008, 3, 360–363. 13. Zhang, Y.; Qin, Y.; Guo, L.; Zhou, Z.; Liang, Z.; Zhang, C.; Guo, H. Isolation of High Quality RNA From Polyporus umbellatus (Pers.) Fries. Electron. J. Biotechnol. 2012, 15. 14. Jaakola, L.; Pirttila, A.M.; Halonen, M.; Hohtola, A. Isolation of High Quality RNA From Bilberry (Vaccinium myrtillus L.). Fruit. Mol. Biotechnol. 2001, 19, 201–203. 15. Chang, S.; Puryear, J.; Cairney, J. A Simple and Efficient Method for Isolating RNA From Pine Trees. Plant Mol. Biol. Rep. 1993, 11, 113–116. 16. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-DeltaDeltaCT Method. Methods 2001, 25, 402–408. 17. Schroeder, A.; Mueller, O.; Stocker, S.; Salowsky, R.; Leiber, M.; et al. The RIN: An RNA Integrity Number for Assigning Integrity Values to RNA Measurements. BMC Mol. Biol. 2006, 7, 3. 18. Zeng, Y.; Yang, T. RNA Isolation From Highly Viscous Samples Rich in Polyphenols and Polysaccharides. Plant Mol. Biol. Rep. 2002, 20, 417. 19. Wang, W.; Stegemann, J.P. Extraction of High Quality RNA From Polysaccharide Matrices Using Cetlytrimethylammonium Bromide. Biomaterials 2010, 31, 1612–1618. 20. Zhao, L.; Ding, Q.; Zeng, J.; Wang, F.R.; Zhang, J.; Fan, S.J.; He, X.Q. An Improved CTAB– Ammonium Acetate Method for Total RNA Isolation From Cotton. Phytochem. Anal. 2012, 23, 647–650. 21. Wang, G.; Wang, G.; Zhang, X.; Wang, F.; Song, R. Isolation of High Quality RNA From Cereal Seeds Containing High Levels of Starch. Phytochem. Anal. 2011, 23, 159–163.
