Biotechnol Lett DOI 10.1007/s10529-014-1752-6

ORIGINAL RESEARCH PAPER

An improved method for extraction of high-quality total RNA from oil seeds Azadeh Rayani • Fatemeh Dehghan Nayeri

Received: 13 October 2014 / Accepted: 11 December 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Seeds of oilseed plants that contain large amounts of oil, polysaccharides, proteins and polyphenols are not amenable to conventional RNA isolation protocols. The presence of these substances affects the quality and quantity of isolated nucleic acids. Here, a rapid and efficient RNA isolation protocol that, in contrast to other methods tested, allows high purify, integrity and yield of total RNA from seeds of sesame, corn, sunflower, flax and rapeseed was developed. The average yields of total RNA from 70 mg oil seeds ranged from 84 to 310 lg with A260/A280 between 1.9 and 2.08. The RNA isolated with this protocol was verified to be suitable for PCR, quantitative real-time PCR, semi-quantitative RT-PCR, cDNA synthesis and expression analysis. Keywords Oilseeds  Polyphenols  Polysaccharides  Quantitative real-time PCR  RNA extraction  Semi-quantitative RT-PCR  Sesame seed Introduction Isolation of high purity and quantity total RNA is a basic requirement for most molecular applications. However, it is often difficult to extract high quality and A. Rayani  F. Dehghan Nayeri (&) Agricultural Biotechnology Department, Faculty of Engineering and Technology, Imam Khomeini International University, Qazvin, Iran e-mail: [email protected]

sufficient quantity of RNA from the seeds of plants due to the high level of various compounds, including polyphenols, polysaccharides, lignin, carbohydrates, and lipids, that readily bind to RNA and cause low yields of it (Dong 1996; Salzman et al. 1999; Ai et al. 2014). Polyphenolic compounds become oxidized and irreversibly bind proteins and nucleic acids (Salzman et al. 1999; Bressan 1999). Polysaccharides coprecipitate or form covalent complexes with nucleic acids (Gehrig et al. 2000; Carra et al. 2007). Although many protocols have been published for the isolation of total RNA from different plant tissues, the majority are not completely satisfying as they may be time-consuming (Yin et al. 2011; Porto et al. 2010), technically complex (Carra et al. 2007; Ren et al. 2008), require ultracentrifugation steps (Carra et al. 2007) are specific to a particular plant species (Ma and Yang 2011). Even some protocols developed by biotechnology companies as high quality and efficiency RNA extraction kits do not always provide the required performance. Most of these commerciallyavailable kits are not suitable for all plant tissues (Xing 1988). Therefore, one RNA isolation method developed for a particular plant is not necessarily suitable for other plants especially for oil seeds that are rich in lipids and proteins (Malnoy et al. 2001; Asif et al. 2000; Iandolino et al. 2004). Since there is no protocol to obtain large quantities of pure RNA from sesame seeds, we report a suitable method that is convenient to isolate RNA from seed tissue of sesame and other oil crops. To our knowledge, this is the first report of a

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highly efficient method to extract RNA from seeds of the sesame plant and then is the foundation for molecular biological research into sesame.

Materials and methods Plant material Seeds of sesame, corn, sunflower, flax and rapeseed were obtained from Seed and Plant Improvement Research Institute in Karaj, Iran. Total RNA extraction Solutions and reagents RNA extraction buffer: 100 mM Tris/HCl (pH 8.0), 50 mM EDTA (pH 8.0), 8 M LiCl, 2 % (w/v) polyvinylpolypyrrolidone (PVP) (Sigma, MW 40000), 2 % (v/v) SDS and 1.5 % (v/v) ß-mercaptoethanol (added just before use); 3 M sodium acetate (pH 5.2); chloroform/isoamyl alcohol (24:1, v/v); phenol/chloroform/ isoamyl alcohol (25:24:1, v/v/v); 2-propanol; ethanol (70 %, v/v); water-saturated acidic phenol (pH 4.5). All solutions were treated with 0.1 % (v/v) diethyl pyrocarbonate (DEPC), except that the Tris buffer and 70 % (v/v) ethanol were prepared with DEPC-treated water.

supernatant was then transferred into a new 2 ml tube and re-extracted with an equal volume of phenol/ chloroform/isoamyl alcohol (25:24:1, v/v), mixed for 2 min and centrifuged at 14,0009g for 10 min at 4 °C. After transferring the supernatant into a new 2 ml tube, an equal vol 2-propanol and 0.1 vol 3 M sodium acetate (pH 5.2) were added, mixed by inversion and then stored at -80 °C for 1 h. Following centrifugation at 14,0009g for 30 min at 4 °C the aqueous phase was discarded and the pellet washed with 200 ll 70 % (v/v) cold ethanol. Air dried pellet was dissolved in 50 ll RNase-free water and 0.3 vol 8 M LiCl and kept overnight at 4 °C. After centrifugation at 14,0009g for 30 min at 4 °C, the precipitate was washed with 70 % (v/v) cold ethanol, air-dried for 10 min and then resuspended in 30 ll RNase-free water and stored at -80 °C. Quality and quantity assessment of total RNA Purity and concentration of RNA samples were evaluated by measuring the A260/A230 and A260/A280 ratios that are indicative of contamination by polyphenols, carbohydrates and proteins. The integrity of extracted RNA was checked on a 1.5 % (w/v) agarose gel stained with ethidium bromide and visualized under UV light. DNase treatment

RNA extraction protocol To extract total RNA, 70 mg dry seeds were frozen in liquid N2, pulverized by grinding with pre-chilled mortar and pestle to a fine powder and added to 2 ml Eppendorf tube containing 1 ml extraction buffer, mixed by gentle shaking or vortexing. [It should be noted here that all glassware (including mortars and pestles) used in the experiment was baked for 2 h at 180 °C. Also, 2 ml Eppendorf tubes and tips were immersed overnight in 0.1 % DEPC treated water and then autoclaved for 30 min.] 500 ll water-saturated acidic phenol was added to the homogenate and mixed for 5 min. Then 250 ll chloroform/isoamyl alcohol (24:1, v/v) was added, mixed well by vortexing for 15 s and centrifuged at 12,0009g for 10 min at 4 °C. The supernatant was pipetted off to a new 2 ml tube, 500 ll volume of chloroform/isoamyl alcohol (24:1, v/v) was added, mixed well by vortexing for 15 s and centrifuged at 12,0009g for 10 min at 4 °C. The

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In order to eliminate genomic DNA from RNA samples, the extracted RNA was treated with RNasefree DNaseI (Fermentas) according to the manufacturer’s instructions. Reverse transcription PCR RNA was reverse transcribed in 20 ll to generate single-stranded cDNA. For this purpose, 1 lg total RNA treated with RNase-free DNaseI was used as a template using oligo(dT) primer (1 lg/ll, Qiagen) for 5 min at 65 °C. Then, reaction mixture was incubated with the AMV reverse transcriptase (200 U/ll, Fermentas) for 60 min at 42 °C. The reaction was inactivated by heating the mixture at 72 °C for 10 min. The 18S rRNA gene (Noguchi et al. 2008) was amplified using cDNA template (1 lg) in 10 ll using PCR master mix in PCR reaction. Reverse transcription PCR reaction was performed in a thermal

Biotechnol Lett

cycler. The initial denaturation step (94 °C for 2 min) followed by 35 cycles of 94 °C for 30 s (denaturing), 55 °C for 30 s (annealing), 72 °C for 15 s (extension) and a final extension period at 72 °C for 5 min. PCR products were separated on a 2 % (w/v) agarose gel and visualized by ethidium bromide staining.

which 35 represented the number of PCR cycles. Primers for housekeeping gene (18S rRNA) were designed by the Oligo 5,v 3.0 software as followed: 50 CGTCCCTGCCCTTTGTACAC-30 (forward) and 50 CGAACACTTCACCGGACCAT-30 (reverse).

Analysis of gene expression by semi-quantitative RT-PCR

Results and discussion Quantity and quality of isolated RNA

The expression level of the three CYP genes including CYP81Q1, CYP81Q2 and CYP81Q3 was measured in seed tissues of two Iranian cultivars by semi-quantitative RT-PCR technique. The PCR reaction was performed using 1/20 of the reverse transcription PCR reaction 20 ll containing 10 pmol specific primers for coding region of the CYP genes. The primers specific to 18S rRNA gene were used as control. PCR was carried out under the following conditions: 2 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 15 s at 72 °C, with a final extension for 5 min at 72 °C. The experiments were repeated three times. Amplification products, 5 ll, were separated on a 1.2 % (w/v) agarose gel and visualized by ethidium bromide staining. Analysis of gene expression by quantitative realtime PCR (qRT-PCR) The expression of the CYP81Q1 gene was analyzed in seed tissues of 10 sesame cultivars by qRT-PCR technique. For this purpose RNA was isolated from these cultivars using our method. Quantitative real-time PCR was performed in 96-well plates on a Bio-Rad iQ5 real-time PCR detection system using SYBR Green reagent (Tiangen, China). Reactions were carried out in 20 ll containing 5 ll template (cDNA), 2 ll 10 pmol/ ll primer mix (forward and reverse) and 10 ll SYBR green master mix. The standard thermal profile was used for all reactions as: 94 °C for 30 s followed by 35 cycles of 94 °C for 10 s, 60 °C for 10 s and 72 °C for 30 s. Dissociation curves were obtained from a thermal melting profile generated under a final PCR cycle of 95 °C for 30 s followed by a constant increase in temperature from 60 to 95 °C. CT values (CTg) were normalized based on the CT value of the 18S rRNA as housekeeping gene (DCT = CTg - CT18SrRNA) and then 35 -DCT values were calculated and plotted, in

The current study presents an optimized method allows efficient isolation of high-quality total RNA from small amounts of the seeds. This method is a modified version of Hou et al. (2011) protocol for RNA extraction from Fritillaria unibracteata that is based on the phenol, PVP and phenol/chloroform/ isoamyl alcohol. In this method, phenolic compounds are bound to phenol and then eliminated by 2-propanol precipitation of the nucleic acids. The solubility of polysaccharides is increased by high LiCl molarity of the extraction buffer and subsequently removed by LiCl precipitation (Dang and Chen 2013; Yin et al. 2011; Ma and Yang 2011; Ren et al. 2008). Our established protocol is fast, simple and efficient that does not require CTAB, Trizol, proteinase K and ultracentrifugation. This protocol was employed for successful RNA extraction from seeds of sesame (Sesamum indicum), corn (Zea mays), sunflower (Helianthus annuus), flax (Linum usitatissimum L.) and rapeseed (Brassica napus). In this study we used several protocols of RNA isolation reported by Ma and Yang (2011), Yin et al. (2011) and Hou et al. (2011), commercial RNA isolation kit (RNX-PLUS kit, CinnaGen, Iran), and also the methods based on Trizol reagent to extract total RNA from oil seeds, but a high yield and quality of total RNA was only obtained with our modified method (Fig. 1; Table 1). RNA isolation procedures are assayed by spectroscopy and electrophoresis based on the quantity, quality and integrity of isolated RNA. Extracted RNA with this method showed the intact sharp 28S and 18S ribosomal RNA (rRNA) bands and the lack of RNA degradation indicated high quality of achieved total RNA (Fig. 1 lanes 1–5) whereas the other procedures led to no results (Fig. 2a–c). Modifications in our protocol were as follows: elimination of the need for lyophilization, elimination

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of ultracentrifugation, no need to use frozen dry seeds free of the seedcase, use of SDS and LiCl into the extraction buffer instead of CTAB, Trizol, guanidium salt and simplification of the extraction step by applying phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v). Differences in the UV absorption spectra maxima of pure nucleic acids (Amax = 260 nm), proteins (Amax =

Fig. 1 Electrophoretic analysis of RNA isolated from oil seeds. The RNA samples were separated on 1.5 % agarose gel containing ethidium bromide and photographed under UV light. M 1 kb DNA marker, lane 1: sesame, lane 2: corn, lane 3: sunflower, lane 4: flax and lane 5: rapeseed

Table 1 Absorbancy ratios and yields of RNA isolated from seeds by our protocol and other methodsa

Method

Our protocol

Kit

Ma and Yang method

Yin et al. method

a

Values are mean ± SD (n = 3) b

lg in 70 mg dry seeds

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280 nm) and polysaccharides (Amax = 230 nm) have been used as a measure of purity in nucleic acid extraction. A ratio (A260/A280) of around 2.0 is generally accepted as pure for RNA. The A260/A230 values for pure nucleic acids are often higher than the respective A260/A280 values. Expected A260/A230 ratios are commonly from 2 to 2.2. In comparison to other methods (Yin et al. 2011; Ma and Yang 2011) and assay kits, the A260/A230 values for all RNA samples isolated by our protocol were higher than 1.85 indicating high purity of them. Also, the A260/A280 ratios ranged from 1.9 to 2.08 representing a low protein contamination (Table 1). The average A260/A230 ratios of oil seeds RNA prepared by other protocols ranged from 1.18 to 1.52, suggesting that RNA samples were contaminated by polysaccharides, proteins, phenol or salts. Furthermore, protein contamination in these samples ranged from 1.32 to 1.65, less than the accepted A260/A280 value of 1.8. On the other hand, RNA samples prepared using the other protocols were low in purity (Table 1). The current method produced the highest RNA yields (84–310 lg), that were 4–8 times more

Plant

Yieldb

Absorbancy ratio A260/A230

A260/A280

Sesame

2.01 ± 0.06

2.08 ± 0.03

310

Sunflower

1.98 ± 0.04

1.90 ± 0.05

208

Corn

2.02 ± 0.04

2.03 ± 0.03

286

Flax

1.90 ± 0.02

2.07 ± 0.08

118

Rapeseed

1.85 ± 0.03

1.95 ± 0.07

84

Sesame Sunflower

1.35 ± 0.11 1.58 ± 0.15

1.38 ± 0.09 1.49 ± 0.11

10 13

Corn

1.68 ± 0.06

1.62 ± 0.05

12

Flax

1.59 ± 0.12

1.59 ± 0.1

15

Rapeseed

1.69 ± 0.08

1.65 ± 0.1

16

Sesame

1.27 ± 0.11

1.37 ± 0.04

70

Sunflower

1.38 ± 0.09

1.32 ± 0.08

36

Corn

1.52 ± 0.1

1.45 ± 0.01

59

Flax

1.43 ± 0.04

1.34 ± 0.1

43

Rapeseed

1.43 ± 0.08

1.53 ± 0.1

89

Sesame

1.38 ± 0.06

1.43 ± 0.05

60

Sunflower

1.43 ± 0.04

1.38 ± 0.05

67

Corn

1.35 ± 0.1

1.32 ± 0.05

39

Flax

1.48 ± 0.1

1.53 ± 0.07

61

Rapeseed

1.35 ± 0.05

1.32 ± 0.06

43

Biotechnol Lett Fig. 2 Electrophoretic analysis of RNA isolated from oil seeds. a Visualization of total RNA isolated from oil seeds by kit protocol (CinnaGen, Iran). b Ma and Yang (2011) method. c Yin et al. (2011) method. The RNA samples were separated on 1.5 % agarose gel containing ethidium bromide and photographed under UV light. M 1 kb DNA marker, lane 1: sesame, lane 2: corn, lane 3: sunflower, lane 4: flax and lane 5: rapeseed

than those by the other protocols (10–84 lg) (Table 1). The differences observed in the average RNA yield of different oil seeds are likely due to different amounts of oil and secondary metabolites including lignin (Porto et al. 2010; Carra et al. 2007). The amount of RNA extracted by the present protocol from 70 mg oil seeds [corn (286 lg), flax (118 lg), rapeseed (84 lg), sesame (310 lg) and sunflower (208 lg)] was higher than the other methods (Table 1). In addition, the RNA yields from the current method were higher than the amount obtained from immature seeds of Jatropha curcas L (282 lg from 500 mg dry seeds) (Sangha et al. 2010).

Fig. 3 Molecular analysis of total RNA isolated from oil seeds. Amplification of a 171 bp fragment of 18S rRNA gene by PCR. M 1 kb DNA marker, lane 1: sesame, lane 2: corn, lane 3: sunflower, lane 4: flax and lane 5: rapeseed

Molecular analysis of isolated RNA by semiquantitative RT-PCR RT-PCR technique is used to monitor the clarity and intactness of ribosomal RNA bands. For this purpose first cDNA was checked using 18S rRNA gene by PCR (Fig. 3). The expression of CYP genes (CYP81Q1, CYP81Q2 and CYP81Q3) was analyzed by semiquantitative RT-PCR in the seeds of two Iranian sesame cultivars (Fig. 4). CYP transcripts were present in both cultivars. The expression of CYP genes was represented as a single band without any smearing indicated the integrity of extracted RNA.

Fig. 4 Expression analysis of CYP genes (CYP81Q1, CYP81Q2 and CYP81Q3) in the seeds of two Iranian sesame cultivars (1 and 2) by semi-quantitative RT-PCR. The expression of CYP genes showed a single band without any smearing indicating the integrity of extracted RNA

Molecular analysis of isolated RNA by quantitative RT-PCR PCR amplification of cDNA obtained from RNA isolated using our method was consistent. The visible

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Biotechnol Lett Fig. 5 a RNA isolated from ten Iranian sesame cultivars using our method. b Melting peaks of a PCR product amplified from 18S rRNA cDNA expressed by seeds. c Expression of CYP81Q1 gene was analyzed in the seeds of ten Iranian sesame cultivars by qRT-PCR technique. CT values (CTg) were normalized based on the CT value of 18S rRNA as housekeeping gene (DCT = CTg - CT18SrRNA) and then 35 -DCT values (35 represented the number of PCR cycles) were calculated and plotted

and distinct bands with the expected size of the 18S rRNA gene fragment were detected (Fig. 5a). Average RT-PCR cycle thresholds (CT) were 17 cycles, and the melting curve was specific with a single peak occurring at about 85 °C (Fig. 5b). As it was illustrated in Fig. 5, Iranian sesame cultivars showed differential expression levels of CYP81Q1 gene that was due to their differences in the sesamin content of seeds (Fig. 5c). This pattern was in accordance with sesamin content of the cultivars (data not shown).

Conclusion We have developed a protocol that is simple and efficient for extracting good quality RNA from oil-rich crops. This method will be useful for extraction of

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high quality and quantity of total RNA that is essential for the success of molecular studies of sesame and other oilseed crops.

References Ai FF, Bin J, Zhang ZM, Huang JH, Wang JB, Liang YZ, Yu L, Yang ZY (2014) Application of random forests to select premium quality vegetable oils by their fatty acid composition. Food Chem 143:472–478 Asif MH, Dhawan P, Nath P (2000) A simple procedure for the isolation of high quality RNA from ripening banana fruit. Plant Mol Biol Report 18:109–115 Carra A, Gambino G, Schubert A (2007) A cetyltrimethylammonium bromide-based method to extract low-molecularweight RNA from polysaccharide-rich plant tissues. Anal Biochem 360:318–320

Biotechnol Lett Dang PM, Chen CY (2013) Modified method for combined DNA and RNA isolation from peanut and other oil seeds. Mol Biol Rep 40:1563–1568 Dong JZ (1996) A reliable method for extraction of RNA from various conifer tissues. Plant Cell Rep 7:516–521 Gehrig H, Winter K, Cushman J, Borland A, Taybi T (2000) An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides. Plant Mol Biol Report 18:369–376 Hou P, Xie Z, Zhang L, Song Z, Mi J, He Y, Li Y (2011) Comparison of three different methods for total RNA extraction from Fritillaria unibracteata, a rare Chinese medicinal plant. J Med Plants Res 5:2835–2839 Iandolino A, Dasilva FG, Lim H, Choi H, Williams L, Cook D (2004) High-quality RNA, cDNA, and derived EST libraries from grapevine (Vitis vinifera L.). Plant Mol Biol Report 22:269–278 Ma X, Yang J (2011) An optimized preparation method to obtain high-quality RNA from dry sunflower seeds. Genet Mol Res 10:160–168 Malnoy M, Reynoird J, Mourgues F, Chevrau E, Simoneau P (2001) A method for isolating total RNA from pear leaves. Plant Mol Biol Report 19:69

Noguchi A, Fukui Y, Iuchi-Okada A, Kakutani S, Satake H, Iwashita T, Nakao M, Umezawa T, Ono E (2008) Sequential glucosylation of a furofuran lignan, (?)-sesaminol, by Sesamum indicum UGT71A9 and UGT94D1 glucosyltransferases. Plant J 54:415–427 Porto B, Magalhaes P, Campos N, Alves J, Magalhaes M (2010) Optimization of RNA extraction protocols using different maize tissues. RBMS 9:189–200 Ren ZK, Yu SL, Yang QL, Pan LJ, Chen MN, Zheng Q (2008) An efficient method of total RNA extraction from peanut seeds. Peanut Sci 37:20–23 Salzman RA, Fujita T, Zhu-Salzman K, Hasegawa PM, Bressan RA (1999) An improved method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol Biol Report 17:11–17 Sangha JS, Gu K, Kaur J, Yin Z (2010) An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L. BMC Res Notes 3:126–134 Xing S (1988) A method for RNA isolation from marine macroalgae. Anal Biochem 174:650–657 Yin D, Liu H, Zhang X, Cui D (2011) A protocol for extraction of high-quality RNA and DNA from peanut plant tissues. Mol Biotechnol 49:187–191

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