A modified one-step procedure for rapid RNA isolation from insect

A modified guanidinium isothiocyanate method was used to extract total RNA from two forest insect species Clostera anastomosis and Saperda populnea. The integrity of RNA was demonstrated by the methods of gel electrophoresis and cDNA analysis. Typical A260/ A280 absorbance ratio of the total RNA was in range of 1.8 to 2.0. The size of double strand cDNAs obtained by RT-PCR was more than 2 kb, which indicated that intact mRNA was obtained. The fragments of β-actin and chitinase gene from the RNA of C. anastomosis were obtained by RT-PCR, which indicated that the RNA could be used for other molecular operation. By this procedure, RNAs could be extracted and analyzed by electrophoresis from at least 8 samples within 4 hours. These results showed that this method was time- and cost-saving and effective.

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.

A Simple and High Quality Method for Isolation and Extraction of Total RNA of Pholiota adipose

Analyzing functional values of RNA using RNA-seq technology is a hot spot of researches. In order to study the medicinal value of Pholiota adipose from the transcriptome level,it is necessary to extract and isolate RNA samples of high purity and high quality P. adipose. In this study,liquid nitrogen grinding Trizol one-step method was used to extract the total RNA of P. adipose. Quality test and statistical comparative analysis were carried out for RNA extract of liquid nitrogen grinding treated and untreated P. adipose. The results showed that the concentration of RNA in the samples treated with liquid nitrogen was much higher than that of the samples without grinding treatment. The OD260/280 of both was about 2,indicating that the purity of RNA was very high. Besides,the ratio of fluorescence intensity of 25 S and 18 S subunit strips of three replicate samples was 1. 8,1. 9,and 1. 9,close to 2,indicating RNA integrity is good. RIN test results of Agilent 2100 were 9. 1,8. 7,and 9. 3,higher than the standard value 6. 8,further proving the integrity. In sum,liquid nitrogen grinding Trizol one-step method is a very simple and efficient method for extracting high quality total RNA of P. adipose.

Isolation of Functional RNA from Heavily Infested, Wilted and Necrotic Leaf Tissues of Tea with High Polyphenol Content

A procedure is described to isolate intact RNA from tissues not previously undertaken-highly infested and wilted apical buds and leaves of tea [Camellia sinensis （L.） O. Ktze.]. The protocol uses a final concentration of 450 mM β mercaptoethanol （βME） and 10% Polyvinylpyrrolidone （PVP） to circumvent problems associated with large amounts of polyphenols, polysaccharides, pigments and other secondary metabolites not easily removed by conventional procedures. Furthermore, the proposed protocol is applicable to normal tissues and other plant tissues with similar stresses, containing compounds that interfere with RNA extractions. Total RNA could be used for downstream applications such as mRNA isolation, reverse transcription, quantitative Polymerase Chain Reaction （PCR）, cDNA library construction and Rapid amplification of cDNA ends （RACE）.