RNA extraction with high quality and quantity is a prerequisite to study analytical methods in plant genetic, molecular biology and related physiological investigations (Gen et al., 2002). These analytical methods like transcriptom analysis by RT-PCR provide simple tools for studying gene expression at the molecular level, segregation of mutation and monitoring the transcription of different type of gene alleles in various plant tissues (Gen et al., 2002; Berendzen et al., 2005).

Also quality and quantity of RNA is essential for ensuring that the sample represents all expressed gene in a cDNA library. However, extracting RNA from plant tissues can be difficult and often require the modification of existing protocols or the development of new procedure (Gasic et al., 2004). Between them, seeds and fruits because of containing large amount of polysaccharides, polyphenol compounds and proteins are one of the most difficult plant tissues for nucleic acid isolation (Gen et al., 2002; Singh et al., 2003).

Common protocols for RNA extraction from seed tissues are tedious and usually result in poor yields. We have systemically tested some protocols for RNA isolation from wheat seeds for the efficiency of integrity and quality of RNA.

In this study, we evaluated a low-cost extraction protocol that has efficiency of integrity and quality of RNA from various wheat seed tissues (embryo, endosperm with aleurone layers and whole seed).

Plant materials: Embryo, endosperm with aleurone layer and whole seed of dormant and after-ripened wheat (Triticum aestivum) cv. RL4137 (a Canadian hard red spring wheat) were used in this research. Wheat seeds were harvested at Zadoks' Growth Stage 92 (Zadoks et al., 1974) grown in the experimental farm at the University of Tehran in 2004. RL4137 expresses high levels of seed dormancy at this stage (Tavakkol Afshari and Hucl, 2002). This experiment was conducted in 2004-2005 in the Plant Molecular Breeding Laboratory, Department of Agronomy and Plant Breeding, University of Tehran. For after-ripening treatments, seeds were kept in germinator at 23°C under dark and dry conditions for six weeks. Following tissues were collected at different experimental treatments; namely: Dormant and after-ripened embryo, dormant and after-ripened endosperm with aleurone layers and dormant and after-ripened of intact seed. The above-mentioned tissues were collected and frozen in liquid nitrogen and kept at -80°C until it is used.

RNA extraction protocol according to Naito et al. (1994): Add 300 μL of EB and 300 mL of PCI to an eppen tube on ice. Cool the mortar and pestle with liquid N2, add 10 seed pods (or 0.05 g of 8DAF siliques) per sample and homogenise very well, adding N2 as necessary to keep sample frozen. Once fully ground, use small spoon to transfer sample to the eppen tube with the buffer. Vortex for 3 min and divide the sample into 2 eppen tubes. Centrifuge for 1 min and collect sup into new eppen. Add an equal volume of PCI and vortex. Centrifuge for 5 min and collect sup into new eppen. Centrifuge again for 5 min and collect sup into new eppen. Add 0.1 vol 3 M NaOAc and 3 vol 100% EtOH, invert 4 times to mix. Place at -80°C (dry ice) for 10 min. Centrifuge for 5 min, discard sup and vacuum dry pellet. Dissolve pellet with 100 μL ddH₂O (may take time). Centrifuge for 10 min and collect sup into new eppen. Add equal volume of 4 M LiCl, invert 4 times to mix. Place on ice on 4°C cold room overnight or -80°C (dry ice) for 1 h.

Next day: Centrifuge for 15 min, discard sup. Small pellet should be visible, be careful as it moves easily. Add 1 mL of 2 M LiCl and carefully invert once to mix. Centrifuge for 2 min and discard sup. Add 1 mL of 70% EtOH and invert once to wash. Centrifuge for 2 min, discard sup and vacuum dry. Dissolve pellet in 5-10 μL ddH2O. Combine the 2 solutions/sample into one eppen. Centrifuge for 5 min and collect sup into new tube. Store at -30°C or lower.

Solutions and reagents: Diethyl Pyrocarbonate (DEPC) treated water was used for all solutions except Extraction Buffer (EB). Extraction Buffer includes; 1M Tris-HCl (MERCK) pH 9.0, 1% SDS. Other used solutions: DEPC water-saturated Phenol+Chloroform–Isoamylalchol (PCI) 25:24:1 V/V, it`s better to mix them just before the extraction, (in Naito et al. protocol phenol was saturated with TE). 3M NaOAC (MERCK) pH 5.6, 4M LiCl (MERCK), 2M LiCl, 100 and 70% EtOH and DEPC- treated water. All the solutions except phenol and chloroform Isoamylalchol were autoclaved.

Experimental protocol for total RNA extraction: This protocol is an improvement and modification of the soybean seed RNA extraction method described by Naito et al. (1994). Our experimental protocol was conducted in four steps in the following pattern:

Step 1: Homogenization

Step 2: Phase separation

Step 3: RNA precipitation

Step 4: RNA recovery

RNA analysis: RNA quantification was performed spectrophotometrically (JENWAY-6405UV/vis Spectrophotometer) at wave lengths of 230, 260 and 280. To confirm the RNA quality, the RNA was electrophoresed on 1.1% agarose gel containing formaldehyde.

RT-PCR analysis: First strand cDNA was synthesized from 0.2 μg of dormant and after-ripened RNA with oligo dT used as a primer and following the RT manufacture`s instruction of Fermentaz Company. A touch down PCR (MgCl₂ 50 mM, dNTPs 10 mM, forward primer 20 μM, reverse primer 20 μM, PCR buffer 10 X, Taq DNA polymerase, all material prepared from Fermentas company and followed the instruction) was performed from 1 μL cDNA by primers which were constructed based on the conserved sequence for wheat MAP Kinases (Sambrook and Russell, 2001). PCR amplification was performed to demonstrate that the RNA could also be used for other analysis (Data about MAP kinases research not shown). For electrophoresis PCR products were loaded on 6% polyacrylamide denaturing gels and stained by silver nitrate method.

Denaturing 94°C 4 min

RL4137 cultivar exhibited a gradual loss of dormancy during after-ripening treatment (Table 1). A six weeks of after-ripening period resulted in seed dormancy loss as demonstrated by higher germination (80%).

Table 1:	Germination percentage of RL4137 cultivar after six weeks of after-ripening

Fig. 1:	The results of Naito RNA extraction protocol on wheat seeds, electrophoresed on 1.1% agarose gel containing formaldehyde. Contamination of DNA and other components like proteins were observed

The Naito et al. (1994) unmodified protocol was used at first for RNA extraction from wheat seeds, but the result was poor in quality because the extracted RNA had contamination of DNA, protein and polysaccharides. It is likely that because of TE saturated phenol and basic pH of extraction buffer DNA also separated by extraction buffer. Based on modified protocol, it is recommended to use DEPC-treated water saturated phenol and lower pH to isolate and purify RNA more efficiently (Fig. 1).

Also method of Chomczynski and Sacchi (1987) was examined in this research, but the results were poor for isolating good quality of RNA from wheat seed (at the end of protocol we obtained an insoluble pellet). Singh et al. (2003) reported several methods for RNA isolating from plant tissue rich in starch (Chomczynski and Sacchi, 1987; Logemann et al., 1987; Gehrig et al., 2000; Salzman et al., 1999; Hosein, 2001), but these methods were not good enough for isolating good quality of RNA from wheat seed.

Fig. 2:	RNA extraction from wheat seed with modified protocol, electrophoresed on 1.1% agarose gel containing formaldehyde. There was no evidence for contamination of DNA and other components

Fig. 3:	RT-PCR results on denaturing polyacrylamide gel electrophoresis, Lane 1: Embryo, Lane 2 and 4: Control samples that their cDNA synthesis were performed without RT, respectively. Lane 3: Endosperm and S. Size marker

High quality RNA with no DNA contaminations was obtained through our modified protocol. This protocol kept the integrity of the RNA while disrupting cells and dissolving cell components. For all RNA samples, distinct 28s and 18s rRNA bands without degradation were observed (Fig. 2). RNA quantification was performed spectrophotometrically at wave length of 260 and 280. RNA samples were diluted in water instead of TE prior to spectrophotometric analysis. The 260/280 OD ratio ranged from 1.9- 2.01 indicating a lack of protein contamination (Table 2). Also 260/230 OD ratio ranged from 2.12-2.35 suggesting less polysaccharides contamination (Table 2).

Moreover, the protocol resulted in high-quality RNA, as evidenced by performing RT-PCR (Fig. 3). After synthesizing sscDNA, MAP kinases fragments were amplified from embryo and endosperm with aleurone layers of wheat with the expected size according to degenerate primers of MAP kinases between 150-300 bp. These MAP kinases fragments had sharper band in after-ripened embryo than endosperm (data not shown). In addition the control samples showed no bands, indicating no DNA contamination. However endosperm tissue produced high quality but low yielded total RNA especially when seeds were dormant (Fig. 4). It was necessary to extract RNA from 600-900 mg of endosperm. Moreover, high volume of extraction buffer was needed.

Table 2:	RNA yield and quantity using spectrophotometric method
D: Dormant, AR: After-ripened, 300 mg material was used for each sample

In dormant state, RNA electrophoresis for the same weight materials of embryo, whole seed and endosperm with aleurone layers showed sharper RNA bands for embryo, endosperm and whole seed, respectively (Fig. 5). In addition embryo had higher RNA content than endosperm at the same material weight in dormant state (Fig. 5).

The results for the same embryo material revealed that after-ripened seed had sharper band of RNA than dormant seeds (Fig. 6). Spectrophotometric results approved that RNA content were more in after-ripened seed than in dormant. Gasic et al. (2004) reported similar results about dormant and active lateral buds showing active lateral buds have more RNA content than dormant ones.

The average extraction time of this protocol was approximately 5-8 h. One of the major disadvantages of this isolation method is that toxic chemicals such as phenol were used and therefore the lab safety should be considered. The results of this method could be useful for seeds and tissues containing high level of protein and polysaccharides.

This study was funded by the grant number 7101027/1/01 from the University of Tehran, Vice Chancellor for Research. We would like to thank Dr. P. Hucl from the University of Saskachewan in Canada for providing Canadian seed cultivar.