Establishment of a rapid, inexpensive protocol for

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Dec 7, 2013 - b Cyprus University of Technology, Department of Agricultural ... In addition, optional treatment with RNase A following initial nucleic acid.

Gene 537 (2014) 169–173

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Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops Anastasis Christou a,1, Egli C. Georgiadou b, Panagiota Filippou b, George A. Manganaris b, Vasileios Fotopoulos b,⁎ a b

Cyprus University of Technology, Department of Environmental Science and Technology, 3603 Lemesos, Cyprus Cyprus University of Technology, Department of Agricultural Sciences, Biotechnology and Food Science, 3603 Lemesos, Cyprus

a r t i c l e

i n f o

Article history: Accepted 30 November 2013 Available online 7 December 2013 Keywords: Genomic DNA extraction Fragaria × ananassa Polyphenols Polysaccharides Protocol Total RNA extraction

a b s t r a c t Strawberry plant tissues and particularly fruit material are rich in polysaccharides and polyphenolic compounds, thus rendering the isolation of nucleic acids a difficult task. This work describes the successful modification of a total RNA extraction protocol, which enables the isolation of high quantity and quality of total RNA from small amounts of strawberry leaf, root and fruit tissues. Reverse-transcription polymerase chain reaction (RT-PCR) amplification of GAPDH housekeeping gene from isolated RNA further supports the proposed protocol efficiency and its use for downstream molecular applications. This novel procedure was also successfully followed using other fruit tissues, such as olive and kiwifruit. In addition, optional treatment with RNase A following initial nucleic acid extraction can provide sufficient quality and quality of genomic DNA for subsequent PCR analyses, as evidenced from PCR amplification of housekeeping genes using extracted genomic DNA as template. Overall, this optimized protocol allows easy, rapid and economic isolation of high quality RNA from small amounts of an important fruit crop, such as strawberry, with extended applicability to other recalcitrant fruit crops. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The isolation of sufficient quantity and high quality nucleic acids is a prerequisite for conducting analytical studies on genetics, molecular biology and other related physiological investigations in plants (Hu et al., 2002). Northern blot hybridization, RT-PCR and microarray tests, employed for gene expression and transcriptomic analysis of plants under a variety of conditions, require RNA of high quality (Berendzen et al., 2005). However, the isolation of functional RNA from certain plant tissues rich in polysaccharides, polyphenolic compounds and proteins is often a time-consuming and tedious task. Polyphenols are known to get readily oxidized to form quinones which in turn can irreversibly interact with proteins and nucleic acids to form high molecular weight complexes that hinder isolation of good quality RNA (Japelaghi et al., 2011). In turn, polysaccharides tend to co-precipitate with nucleic acids in low ionic strength buffers (Wang and Stegemann, 2010). In addition, these co-precipitated compounds severely restrict RNA reverse

Abbreviations: EB, extraction buffer; PCI, phenol: chloroform: isoamyl alcohol; RT-PCR, reverse transcription polymerase chain reaction; SDS, sodium dodecyl sulfate; NaOAC, sodium acetate. ⁎ Corresponding author. Tel.: +357 25002418; fax: +357 25002632. E-mail address: [email protected] (V. Fotopoulos). 1 Present address: Agricultural Research Institute, 1516 Nicosia, Cyprus. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.11.066

transcriptase and DNA polymerase functionality, as well as DNA restriction endonuclease activities, and absorbance-based quantification assays (Moser et al., 2004). Furthermore, stressful conditions for plant growth may further encumber the isolation of high quality RNA, since polyphenols and polysaccharides are bioaccumulated in plant tissues under adverse environmental stimuli (Chang et al., 1993). Therefore, many protocols, along with their modifications, have been published over the years for nucleic acid isolation from recalcitrant plants (Berendzen et al., 2005; Henderson and Hammond, 2013; Hu et al., 2002; Samanta et al., 2011; Sharma et al., 2003; Smart and Roden, 2010; Wang and Stegemann, 2010). However, the majority of them pose certain limitations to researchers as they are time consuming (Moser et al., 2004), tissue-specific (Asif et al., 2000) or technically complex (Wang and Stegemann, 2010). In recent years, strawberry has received particular attention from the research community due to the dietary value of its fruits and the economic importance of its cultivation (Tulipani et al., 2008). Strawberry tissues are rich in polyphenolic compounds and polysaccharides (Asami et al., 2003); thus, the isolation of good quantity and quality nucleic acids from its tissues is often challenging. Several protocols have been used for RNA isolation from strawberry plant tissues, using phenol (Mazzara and James, 2000), hot borate (Martínez and Civello, 2008), or cetyltrimethylammonium bromide (CTAB) (Palomer et al., 2006) in the isolation procedure. The current study presents an

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optimized protocol for the isolation of high quality total RNA from small amounts of strawberry leaf, root and fruit tissue, suitable for downstream molecular applications. This procedure represents a significant improvement in terms of time and amount of tissue needed in comparison with established extraction protocols, requiring ca. 3 h (including nuclease treatment) and 100 mg of tissue for successful completion. The protocol presented herein is based on a modified version of the SDS/PCI method of Mortaji et al. (2008) for RNA extraction from wheat seeds. For comparative purposes, an established protocol for RNA extraction from strawberry leaves and petioles using LiCl and sodium dodecyl sulfate (SDS) in the extraction buffer (Mazzara and James, 2000), as well as a commercially available, ready-to-use reagent (TRIzol® Reagent, Invitrogen) commonly used for nucleic acid extraction have also been tested. The proposed protocol was subsequently employed for successful RNA extraction from other fruits such as olive and kiwifruit. 2. Materials and methods 2.1. Plant material Fully expanded leaves, roots and fruit were sampled from sixmonth-old strawberry (Fragaria × ananassa) plants grown hydroponically in a constantly aerated half-strength Hoagland nutrient solution in 15 l pots. Conditions in the growth room were 16 h photoperiod (250 μmol m−2 s−1), 23 °C/20 °C day/night temperature and 65% relative humidity. Olive fruit (Olea europaea) and kiwifruit (Actinidia deliciosa) were harvested from commercial orchards at maturity stage. All samples were flash-frozen in liquid nitrogen and stored at −80 °C until needed. 2.2. Solutions and reagents The RNA extraction buffer (EB) was similar to that proposed by Mortaji et al. (2008), with the exception of the use of 0.5 M instead of 1 M Tris–HCl (Merck) pH 9, along with 1% (w/v) sodium dodecyl sulfate (SDS). Other solutions used were saturated phenol: chloroform: isoamyl alcohol (PCI) 25:24:1 (v/v), 3 M sodium acetate (NaOAC) (Merck) pH 5.6, as well as 100 and 70% (v/v) ethanol. All experiments were carried out in triplicate using independent pools of tissue samples. 2.3. Nucleic acid isolation procedure Nucleic acid isolation was carried out based on a protocol proposed by Mortaji et al. (2008) for RNA extraction from wheat seeds, following several major modifications: 1. Initially, plant tissue samples were ground into fine powder in a prechilled pestle and mortar under liquid nitrogen. Subsequently, 100 mg of homogenate sample (instead of 300 mg) was transferred to 2 ml tubes containing 1 ml ice-cold EB (0.5 M Tris–HCl pH 9, 1% (w/v) SDS), instead of 1.5 ml pre-warmed EB. Sample was agitated immediately and 1 ml of PCI was added to the tube. The sample was gently agitated for an additional 2 min to form an emulsion and then centrifuged at 14,000 ×g for 5 min at 4 °C for phase separation. 2. The upper aqueous phase (~800 μl) was collected into a fresh chilled 2 ml tube and equal volume of PCI was added and gently agitated. Sample was centrifuged at 14,000 ×g for 7 min at 4 °C. 3. The aqueous phase (~ 600 μl) was collected into fresh chilled tube and the sample was re-extracted with an equal volume of PCI, agitated and centrifuged as previously described. 4. The upper aqueous phase (~ 400 μl) was carefully transferred to a fresh chilled tube and centrifuged at 16,000 ×g for 7 min at 4 °C to remove traces of phenol.

5. The supernatant was collected into a fresh chilled tube, where 0.1 volume 3 M NaOAC (pH 5.6) and 1 volume (instead of 3) 100% (v/ v) ethanol were added, mixed by inversion and subsequently incubated at −80 °C for 20 min for nucleic acid precipitation. 6. Sample was then centrifuged at 16,000 ×g for 8 min at 4 °C, supernatant was discarded and 1 ml 70% (v/v) ethanol was added to the tube, centrifuged at 16,000 ×g for 3 min at 4 °C and supernatant discarded again. 7. Nucleic acid pellet was air-dried at room temperature (or heated at 50 °C for 2–3 min), and finally dissolved in 20–30 μl ddH2O, depending on the pellet size. Alternative isolation protocols included the protocol described by Mazzara and James (2000) and the ready-to-use TRIzol® Reagent (Invitrogen, USA) following the manufacturer's instructions. 2.4. Nucleic acid quantification and quality control The nucleic acid quantity and purity were determined spectrophotometrically by measuring absorbance ratios A260/A230 and A260/A280, indicative of contamination by polyphenols/carbohydrates and proteins, respectively, using the NanoDrop (ND-1000, Thermo Scientific, Delaware, USA). The integrity of extracted nucleic acids was verified by running 1 μg sample in a 1.5% (w/v) agarose gel, stained with GelRed™ (Biotium, Inc., USA).

2.5. DNase and RNase treatment The DNase I Set (Macherey-Nagel GmbH & Co., Germany) was used to purify RNA from DNA-contaminated samples according to manufacturer's protocol. In turn, in order to eliminate RNA from extracted samples for genomic DNA isolation, nucleic acid samples were incubated with RNase A (Macherey-Nagel GmbH & Co., Germany) according to the manufacturer's instructions.

2.6. Reverse transcription (RT) and polymerase chain reaction (PCR) For first-strand cDNA synthesis, 1 μg total RNA was reversed transcribed using the Primescript 1st Strand Synthesis kit, as per manufacturer's instructions (Takara Bio Inc., Japan). The cDNA synthesized was then diluted 1:10 (v/v) with DEPC-treated water and stored at − 20 °C for PCR amplification. The strawberry glyceraldehyde 3phosphate dehydrogenase (FaGAPDH) gene (Miyawaki et al., 2012) was amplified using both cDNA and genomic DNA (after RNase treatment) templates (1 μg) in a 10 μl reaction mix containing Taq DNA polymerase (Invitrogen Inc.). The initial denaturation step (95 °C for 5 min) in PCR reaction was followed by 40 cycles of 95 °C for 30 s, 56 °C for 45 s and 72 °C for 45 s, and a final extension period at 72 °C for 10 min. The amplified product was visualized by gel electrophoresis in a 1.5% (w/v) agarose gel. Similarly, primers for kiwifruit and olive fruit were used in identical cycles for the amplification of actin (AdACT; Yin et al., 2010) and ubiquitin (OeUBQ2; Hernández et al., 2009) housekeeping genes, respectively. Oligonucleotide sequences that were used in this study are listed in Table 1. Table 1 Oligonucleotides primers used for PCR and RT-PCR. Gene

Primer Primer sequence

FaGAPDH For Rev AdACT For Rev OeUBQ2 For Rev

Reference

5′-TCCATCACTGCCACCCAGAAGACTG-3′ Miyawaki et al. (2012) 5′-AGCAGGCAGAACCTTTCCGACAG-3′ 5′-TGCATGAGCGATCAAGTTTCAAG-3′ Yin et al. (2010) 5′-TGTCCCATGTCTGGTTGATGACT-3′ 5′-AATGAAGTCTGTCTCTCCTTTGG-3′ Hernández et al. (2009) 5′-AAGGGAAATCCCATCAACG-3′

A. Christou et al. / Gene 537 (2014) 169–173

3. Results and discussion Several total RNA extraction protocols exist that can be applied in strawberry tissues. These, however, are subject to important limitations: many reports utilize expensive commercial extraction kits using columns (Chai et al., 2013; Lin et al., 2013), others follow lengthy protocols (up to 3 days) optimized for other plant species that require large amounts of starting material (Chen et al., 2011; Pombo et al., 2011), while other strawberry-based protocols are optimized for specific tissues (i.e. leaves and petioles; Mazzara and James, 2000). The current study describes a rapid, efficient and reliable protocol that allows the extraction of high quality total RNA from strawberry plant tissues as well as other fruit crops rich in polysaccharides and polyphenolic compounds. In contrast to the other methods tested, the RNA prepared by this protocol was of high quality and quantity, and was successfully used for downstream applications such as RT-PCR and qRT-PCR. Furthermore, optional treatment with RNase A following initial nucleic acid extraction provided sufficient amounts of genomic DNA which could be successfully used for subsequent PCR analyses. The proposed protocol represents a marked improvement in terms of starting material required, as 100 mg of homogenate is needed instead of 300 mg for successful completion of the protocol by Mortaji et al. (2008) or 1–3 g required by Mazzara and James (2000). This effectively allows for a 60% reduction of the space needed for sample storage in −80 °C refrigerators, which could be particularly important for large scale experiments. In addition, the present protocol can be carried out within approximately 3 h by a moderately-experienced researcher, in comparison with ~ 6 h required for the protocol by Mortaji et al. (2008) or the overnight incubation needed in the Mazzara and James (2000) procedure. It should be noted that the widely popular hot borate method used in strawberry studies can take up to 3 days (Wan and Wilkins, 1994). In addition, lower amounts of reagents are used throughout the procedure, thus contributing to further lowering of costs. Nucleic acids isolated from strawberry tissues following the proposed modified protocol were compared, in terms of quantity and quality, with those isolated from the protocol proposed by Mazzara and James (2000) and the ready-to-use TRIzol® Reagent (Invitrogen). As shown in Table 2, total nucleic acid quantity was the highest using the TRIzol® Reagent according to spectrophotometric quantification, followed by similar range amounts using the proposed protocol and Table 2 Spectrophotometric determination of extracted nucleic acid quantity and quality from strawberry plant tissues following the proposed protocol of Mazzara and James (2000), and TRIzol® Reagent. Proposed protocol was subsequently used on other recalcitrant fruit tissues (olive, kiwifruit). Protocol used

Tissue

Nucleic acids yield (μg/100 mg FW)

A260/A280 ratio

A260/A230 ratio

TRIzol® Reagent

Strawberry leaf Strawberry root Strawberry fruit Strawberry leaf Strawberry root Strawberry fruit Strawberry leaf Strawberry root Strawberry fruit Kiwifruit Olive fruit

25.8

1.26

0.59

97.1

1.12

1.13

37.9

1.26

0.58

11.1

1.12

0.91

6.1

1.16

0.59

4.8

1.13

0.52

7.9

1.93

1.47

7.8

1.87

1.34

9.7

1.69

0.86

14.7 9.4

1.80 2.00

1.34 2.13

TRIzol® Reagent TRIzol® Reagent Mazzara and James (2000) Mazzara and James (2000) Mazzara and James (2000) Current proposed Current proposed Current proposed Current proposed Current proposed

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the one used by Mazzara and James (2000). However, nucleic acid purity indicated by A260/A280 absorbance ratios was below 1.26 and 1.16 for Trizol and LiCl-based protocols, respectively, indicative of protein contamination (Sambrook and Russell, 2001), while the respective pellets were viscous, yellow to brown in color and water-insoluble. Contrarily, extractions with our novel protocol resulted in A260/A280 ratios ranging between 1.97 and 1.67. In a similar manner, A260/A230 absorbance ratios indicative of polyphenol and polysaccharide contamination were the highest (ranging between 2.13 and 0.86) following extraction with the proposed protocol, suggesting lower levels of contamination compared with the other assessed protocols. As expected, fruit tissues displayed lower purity levels due to high concentrations of polysaccharides and phenolic compounds and low concentrations of RNA molecules, resulting in low recovery or difficulty in quantifying the sample (Davis et al., 2006). The integrity of the isolated nucleic acids from the three procedures tested was subsequently visualized on 1.5% agarose gels. As evidenced in Fig. 1, nucleic acids extracted with the TRIzol® Reagent protocol were fully degraded, suggesting that the high yields shown by spectrophotometry were likely the result of false measurements of contaminants, as polysaccharides and polyphenolic compounds often coprecipitate and contaminate the nucleic acids during the extraction, thereby affecting both the quality and quantity of isolated nucleic acids (Asif et al., 2000). Nucleic acids extracted from strawberry tissues following the Mazzara and James (2000) protocol, as well as the one proposed in this report, revealed the simultaneous presence of both intact ribosomal RNA and genomic DNA bands. However, RNA extracted with either Trizol or the LiCl-based protocol could not be used in downstream applications, since the amplification of FaGAPDH from these templates failed (data not shown), probably due to high amounts of inhibitory polysaccharides (Demeke and Adams, 1992) and/or polyphenols (Moreira, 1998). Treatment with DNase 1 successfully eliminated contaminating genomic DNA from all samples following the proposed protocol, as evidenced by purified RNA agarose gel electrophoresis (Fig. 2A). Complete digestion of genomic DNA following DNase 1 treatment was confirmed carrying out PCR with purified as template, which yielded no products (data not shown). In addition, the quantity and quality of total RNA were satisfactory, yielding 2.21–4.01 μg/100 mg fresh weight (FW), while the corresponding A260/A280 and A260/A230 ranged between 1.98–1.78 and 2.29–1.04 respectively (Table 3). Moreover, reverse transcription of DNase 1-treated RNA isolated following the modified protocol and subsequent PCR using cDNA as template revealed that the isolated RNA was of high quality, since FaGAPDH gene fragments were successfully amplified (Fig. 3A). The proposed protocol has already been successfully employed for the isolation of RNA and subsequent real-time RT-PCR gene expression analysis from hydroponicallygrown strawberry plants under osmotic stress (Christou et al., 2013). Fruit tissues are notoriously difficult for nucleic acid extraction as they have high concentrations of contaminating compounds and low concentrations of RNA molecules (Davis et al., 2006). The modified protocol can also be successfully applied for RNA extraction from other recalcitrant fruit tissues such as olive and kiwifruit, thus providing a useful tool for molecular studies focusing on fruit crops. The protocol was also successfully employed using grapes as starting material (data not shown), therefore potentially having a very wide range of applicability. Spectrophotometric quantification of total RNA yield following DNase 1 treatment of nucleic acids extracted revealed even higher efficiency compared with yields from strawberry tissues, resulting in 3.71 and 4.01 μg/100 mg FW from kiwifruit and olive fruit, respectively (Table 3). The integrity of the isolated RNA visualized on 1.5% agarose gels, revealing the presence of intact ribosomal RNA bands (Fig. 2A). Similarly, RNA purity indicated by A260/A280 absorbance ratios was 1.89 and 1.83 for kiwifruit and olive, while A260/A230 absorbance ratios were 1.25 and 2.29, suggesting high purity levels (Table 3). Most importantly, RNA isolated following the modified protocol could be

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M

1

2

3

4

5

M

6

7

8

9

10

11

Fig. 1. Agarose gel (1.5% w/v) electrophoresis of nucleic acids isolated using TRIzol® Reagent, Mazzara and James, 2000, and proposed protocol. M: GeneRuler 1 kb DNA ladder, 1–3: strawberry fruit (1), leaves (2) and roots (3) using TRIzol® Reagent, 4–6: strawberry fruit (4), leaves (5) and roots (6) following Mazzara and James (2000) protocol, 7–9: strawberry fruit (7), leaves (8) and roots (9) following the proposed protocol, 10: kiwifruit, 11: olive fruit.

(Fig. 3B). These results suggest that the proposed protocol has high potential to also be used (following slight modifications) for rapid isolation of genomic DNA, which is of sufficient quantity and quality for PCR analyses and could therefore be utilized for quick screening of large numbers of transformants etc.

successfully used for downstream RT-PCR applications, as revealed by housekeeping gene (AdACT for kiwifruit and OeUBQ2 for olive) amplification (Fig. 3A), while the isolated material was also successfully used in sensitive real-time RT-PCR gene expression analyses using material from both fruit crops (data not shown). The proposed protocol also provides the versatility to be used for genomic DNA isolation, following RNase A treatment after initial nucleic acid extraction. In regard with purity and yield, genomic DNA yielded from strawberry tissues and other fruit samples ranged between 9.42 and 2.71 μg/100 mg FW, whereas A260/A280 and A260/A230 ratios ranged between 1.70–1.18 and 1.02–0.38 (Table 3), suggesting contamination with proteins, polysaccharides and polyphenolic compounds. However, agarose gel electrophoresis of isolated genomic DNA revealed intact bands of high molecular weight (Fig. 2B), while FaGAPDH fragments amplified from purified genomic DNA made apparent that it is of sufficient quality and functionality for downstream PCR applications

A)

M

1

2

3

4

4. Conclusion The proposed protocol offers an efficient, economic and rapid (can be completed within ~ 3 h by a moderately experienced worker) procedure that can be used by molecular biology laboratories for the reliable isolation of RNA from strawberry plant tissues, suitable for an array of downstream applications. Furthermore, the method can be applied to other recalcitrant plant species displaying high amount of secondary metabolites, while it can also be modified to provide sufficient quality amounts of genomic DNA extraction for

5

B)

M

1

2

3

4

Fig. 2. Agarose gel (1.5% w/v) electrophoresis of total RNA samples purified after incubation with DNase I (A), and genomic DNA samples after incubation with RNase A (B), after initial nucleic acid extraction following the proposed protocol. A) M: GeneRuler 1 kb DNA ladder, 1: strawberry fruit, 2: strawberry leaves, 3: strawberry roots, 4: kiwifruit, 5: olive fruit. B) M: GeneRuler 1 kb DNA ladder, 1: strawberry leaves, 2: strawberry roots, 3: kiwifruit, 4: olive fruit.

A. Christou et al. / Gene 537 (2014) 169–173 Table 3 Spectrophotometric determination of total RNA and genomic DNA quantity and quality from strawberry plant tissues and other recalcitrant fruit tissues (olive, kiwifruit), following incubation of nucleic acids (extracted with proposed protocol) with DNase I and RNAse A, respectively. Tissue

RNA yield (μg/100 mg FW)

DNA yield (μg/100 mg FW)

A260/A280 ratio

A260/A230 ratio

Strawberry leaf Strawberry root Strawberry fruit Kiwifruit Olive Strawberry leaf Strawberry root Strawberry fruit Kiwifruit Olive

2.21 3.49 2.37 3.71 4.01 – – – – –

– – – – – 6.93 9.42 4.54 2.71 3.44

1.98 1.80 1.78 1.89 1.83 1.53 1.32 1.18 1.42 1.70

1.81 1.66 1.04 1.25 2.29 0.75 0.65 0.38 0.43 1.02

PCR analysis following RNAse A incubation of the initial extracted nucleic acids. Conflict of interest All authors involved declare no conflict of interest.

A) RT-PCR analysis M

1

M

1

2

3

4

5

6

7

8

B) gPCR analysis 2

3

4

5

6

7

Fig. 3. Agarose gel (1.5% w/v) electrophoresis of housekeeping genes amplified with RTPCR using cDNA (A), and PCR using genomic DNA (B), following the proposed modified protocol. A) M: GeneRuler 1 kb DNA Ladder, 1–3: FaGAPDH amplified from strawberry fruit (1), leaves (2) and roots (3), 4: FaGAPDH negative control, 5: AdACT amplified from kiwifruit, 6: AdACT negative control, 7: OeUBQ2 amplified from olive, 8: OeUBQ2 negative control. B) M: GeneRuler 1 kb DNA Ladder, 1–2: FaGAPDH amplified from strawberry leaves (1) and roots (2), 3: FaGAPDH negative control, 4: AdACT amplified from kiwifruit, 5: AdACT negative control, 6: OeUBQ2 amplified from olive fruit, 7: OeUBQ2 negative control.

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