BENCHMARKS -70°C. This protocol can also be used Rapid method for high-quality RNA isolation for seed tissue above 1 g by adjusting from seed endosperm containing high levels of the volumes of buffers accordingly. All the solutions were prepared from 0.1% starch diethylpyrocaborate (DEPC)-treated water and powder stocks. Glassware Zhiwu Li and Harold N. Trick used in this protocol was cleaned with Kansas State University, Manhattan, KS, USA detergent, filled with 0.1% DEPC, incubated at 37°C overnight, and then BioTechniques 38:872-876 (June 2005) autoclaved for 30 min. Standard RNA extraction methods using GITC-phenol-chloroform (1), RNeasy kit, or TRIzol reagent failed to produce satisfactory RNA when attempting to extract RNA from Isolation of high-quality RNA from mg seeds were ground to a fine powder starch-rich seed species such as wheat, plant seeds is very critical for seed- in liquid nitrogen with prechilled rice, and maize (data not shown). A specific gene analysis. However, seed mortar and pestle. The flour sample large starch percentage causes sample endosperm contains very high levels was then transferred into a prechilled solidification when RNA extraction is of starch, which cause the solidifi- 1.5-mL RNase-free microcentrifuge attempted with buffers from the above cation of samples in the guanidine tube. A 400-µL extraction buffer I [100 mentioned protocols. In addition, isothiocyanate (GITC)-based RNA mM Tris, pH 8.0, 150 mM LiCl, 50 mM starch tended to co-precipitate with the extraction buffers, such as GITC- EDTA, 1.5% sodium dodecyl sulfate RNA pellet, making it very difficult phenol-chloroform buffer (1), TRIzol® (SDS), 1.5% 2-mercaptoethanol] to redissolve the RNA in water. reagent (Invitrogen, Carlsbad, CA, aliquot was immediately added to the Although a seed flour sample could USA) and the RNeasy® kit (Qiagen, seed powder. After mixing the content be easily disrupted in phenol-SDS Valencia, CA, USA). These current with vigorous vortex mixing, 250 µL RNA extraction buffer when a standard RNA small- and large-scale extraction phenol-chloroform mixture (1:1, pH phenol-SDS protocol was used (2), this protocols typically either fail to yield 4.7) were added, and the samples were protocol failed to produce high-quality RNA or result in reduced yields with mixed well by inversion. Samples RNA due to the serious degradation poor quality. Tissue with a high level were then centrifuged immediately of the RNA as indicated by the degra- of starch can also hinder resuspension at 13,000× g for 15 min at 4°C. The dation of 28S and 18S ribosomal RNA of precipitated RNA or contaminate upper aqueous phase (around 250 µL) (rRNA) as well as a smear of smaller the RNA pellet by co-precipitation was carefully transferred to a new 1.5- sized RNAs (Figure 1). The quality (2). Although several protocols were mL tube containing 250 µL extraction of RNA produced by the phenol-SDS developed to remove polysaccharide buffer II [70% guanidinium sulfate (w/ method was not sufficient for reverse and phenolics contamination from v), 0.75 M sodium citrate, 10% lauryl- transcription PCR (RT-PCR) analysis plant RNA (3–5), most of the existing sarcosine, 2 M sodium acetate, pH 4.0]. (data not shown). protocols are time-consuming and Samples were mixed by gentle inversion The new protocol reported here labor-intensive. Additionally, there are and incubated at room temperature for resulted in the rapid isolation of no satisfactory protocols for isolating 10 min. After the incubation, 200 µL high-quality RNA from starchy seed RNA from starch rich seeds, such chloroform-isoamyl alcohol (24:1) samples of 50–100 mg. The quality of as wheat, rice, and maize, using <1 were added, and the samples were then the RNA prepared by this method was g of seed material. In this report, we centrifuged at 13,000× g for 15 min demonstrated by intact sharp 28S and developed a rapid method for extracting at 4°C. To the recovered supernatant 18S rRNA bands and the lack of RNA high-quality RNA from wheat, rice, (around 450 µL), 300 µL isopropanol, degradation on agarose gels (Figure and maize seed endosperms. It can be and 250 µL 1.2 M sodium chloride 1). We have successfully used this used to isolate seed-derived RNA in were added. The samples were then RNA for both RT-PCR and Northern both small- and large-scale extraction mixed by inversion and put on ice for blot analyses (data not shown). In protocols, and it directly overcomes the 15 min. The sample was centrifuged at this protocol, the following steps and problems of solidification of samples in 13,000× g for 15 min at 4°C, then the rationale were used: (i) Extraction extraction buffer, starch contamination, supernatants were discarded, and the buffer I, which contains SDS, effec- and starch co-precipitation. RNA pellets were washed carefully tively dissolved seed sample containing Wheat (Triticum aestivum L.) seeds with 400µL 70% ethanol. The RNA high concentrations of starch. Thus, “Bobwhite” were collected around 20 pellets were then dried for 15–20 the problem of sample solidification days post-anthesis. Rice (Oryza sativa min at room temperature in a laminar due to excess starch in the seed was L.) and maize (Zea mays L.) seeds were flow hood and were resuspended in resolved. Solidification could not be collected 23 days post-anthesis. For the the appropriate volume of RNase avoided when using other TRIzol- or new RNA extraction procedure, 50–100 free water (e.g., 50 µL) and stored at GITC-containing buffers. The addition 872 BioTechniques Vol. 38, No. 6 (2005) BENCHMARKS Table 1. Yield and Purity of Total RNA Prepared by the New Protocol and Standard Phenol-SDS Method Evaluated by UV Light Absorption Spectra and Ratios of A260/A280 and A260/A230 a a b Seed A260/A280 A260/A230 RNA Yield Species (µg/100 mg FWc) Phenol-SDS New Protocol Phenol-SDS New Protocol Phenol-SDS New Protocol Wheat 1.54 ± 0.25 1.98 ± 0.02 0.34 ± 0.10 1.85 ± 0.03 18.3 ± 3.2 48.5 ± 4.1 Rice 1.69 ± 0.10 1.95 ± 0.02 0.77 ± 0.09 2.01± 0.02 25.7 ± 2.5 50.3 ± 3.2 Maize 1.57 ± 0.14 1.98 ± 0.03 0.90± 0.05 1.89 ± 0.03 28.5 ± 1.9 51.5 ± 2.8 SDS, sodium dodecyl sulfate; FW, fresh weight. a Ten independent RNA extraction replicas of each species that were measured for analysis values of A260/A280 or A260/A230 are mean ± SD. bRNA yields using the phenol-SDS protocol were significantly different from yields with the new protocol at the 95% confidence level. of 2-mercaptoethanol aided in RNase A260/A280 and A260/A230 ratios (6), ratio at A260/A230 of wheat, rice, and and oxidation protection. (ii) Acid which ranged from 1.96 to 2.00 and maize seed RNA prepared by the phenol-chloroform (1:1, pH 4.7) was 1.80 to 1.90, respectively. In addition, standard phenol-SDS protocol ranged used to promote cell lysis and for DNA high purity RNA was also prepared by from 0.34 to 0.90, far less than the and protein separation. After phenol- this protocol from rice and maize seeds. accepted A260/A280 value of 1.80. These chloroform extraction, the bulk of the A260/A280 and A260/A230 ratios of rice results indicated that RNA prepared starch was removed. (iii) A GITC- seed RNA prepared by our protocol by phenol-SDS was low in purity and based extraction buffer II ensured ranged from 1.93 to 1.97 and 1.99 to that the purity was highly variable as RNase inhibition. Extraction buffer II, 2.03, respectively, and A260/A280 and implied by the high standard errors of which was added immediately after A260/A230 ratios of maize seed RNA A260/A280 or A260/A230 (Table 1). the phenol-chloroform extraction, prepared by our protocol ranged from In conclusion, we report a new, prevented the degradation of RNA. 1.95 to 2.01 and 1.86 to 1.92, respec- efficient, and reliable RNA extraction Guanidinium sulfate, a strong protein tively (Table 1). The average RNA method for wheat seed endosperm. denaturant, may be replaced by other yields from 100-mg seed samples This method can also be used for RNA guanidine salts such as in TRIzol. of wheat, rice, and maize were 48.5, extraction from other plant seeds with (iv) High concentration of salt (1.2 M 50.3, and 51.5 µg respectively (Table high starch content, such as rice and sodium chloride) and low isopropanol 1), which were approximately double maize. By this method, the isolated concentration (30%) were added into the corresponding RNA yields from RNA from 50–100 mg seed tissue the RNA precipitation step to provide the phenol-SDS protocol. The RNA was of high quality and quantity, and maximum starch solubility. This purity from a 100-mg seed sample was it could be used for RT-PCR analysis modification effectively maintained acceptable for RT-PCR, cDNA library and Northern blot analysis. Therefore, carbohydrates in a soluble form, while construction, or Northern blot analysis. this method would be especially useful helping to precipitate the RNA in On the other hand, the average A260/ for rapid production of RNA for gene high purity. With little carbohydrate A280 of wheat, maize, and rice seed expression analysis of plant tissue with impurities, the RNA pellet was easily RNA prepared by the standard phenol- high starch content.