Description
In recent years, interest in small RNA, such as siRNA and miRNA which are related to research of gene regulation, has expanded. There are many commercial kits for total RNA preparation, but most of these are focused on preparation of large RNA longer than 200 nucleotides. Because both siRNA and miRNA are between 15 ~ 30 nucleotides in length, the need of specially optimized kit for small RNA (< 200 nucleotides) is growing rapidly. Hybrid-RTM miRNA is designed for purification of large and small RNA separately from culture cells or animal tissues and co-purification in a single tube is also available by modified protocol. This kit utilizes the lysis method of RiboExTM which has a powerful ability of lysis and the purification method based on glassfiber membrane technology. Samples are homogenized in RiboExTM, a monophasic solution containing phenol and guanidium salt, which rapidly lyse cells and inactivates nucleases. Addition of chloroform brings about a separation of the lysate into aqueous and organic phases. Total RNA locates in the aqueous phase while DNA and protein remain in the interphase and organic phase. Large and small RNA in the aqueous phase is selectively bound to column type B and type W respectively. The column type B selectively adsorbs the RNA larger than 200 nucleotides in length, while the column type W specifically holds the RNA smaller than 200 nucleotides in length.To purify large RNA, the aqueous phase is mixed with ethanol and the mixture is applied to a column type B. After centrifugation, large RNA is bound to membrane and the mixture containing small RNA goes into collection tube through the membrane. The membrane is washed away by two wash buffer (SW1 and RNW) and purified large RNA is eluted from the membrane by RNase-free water. To purify small RNA, the pass-through come from the binding of large RNA is mixed with ethanol and then applied to a column type W. After washing with buffer RBW and RNW, small RNA is eluted by RNase-free water. The procedure of Hybrid-RTM miRNA takes only 30 minutes for complete preparations of pure RNA. The purified RNA is suitable for the isolation of Poly A RNA, northern blotting, dot blotting, in vitro translation, cloning, RT-PCR, RPA and other analytical procedures.
Features and Benefits
¦ Preparation time : ~ 30 minutes
¦ Stable and consistent yield
¦ High purity and yield
¦ Perfect separation of small RNA fragment
¦ Sample size : Up to 50 mg tissue or up to 1 x 107 cultured cells
¦ Recovery range : Large RNA : > 200 nucleotides
Small RNA : < 200 nucleotides
¦ Instant use : No need of additional materials
¦ No ethanol precipitation
¦ No Genomic DNA contamination
¦ Ready for use in northern blotting, dot blotting, in vitro translation, cloning, RT-PCR, RPA and other
analytical procedures
Procedure
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