Stored at 2-8ºC
Reagents required, but not supplied: ·
2. Isopropyl alcohol
3. 75% Ethanol (in RNA-free water)
4. RNase-free water or 0.5% SDS solution [To prepare RNase-free water, draw water into RNase-free glass bottles. Add diethylpyrocarbonate (DEPC) to 0.01% (v/v). Let stand overnight and autoclave. The SDS solution must be prepared using DEPC-treated, autoclaved water.]
WARNING: Toxic in contact with skin and if swallowed. Causes burns. After contact with skin, wash immediately with plenty of detergent and water. If you feel unwell, seek medical advice (show label where possible). Phenol (108-95-2) and other Components (NJTSRN 80100437-5000p).
TRIpure Reagent is a ready-to-use reagent for the isolation of total RNA from cells and tissues. The reagent, a mono-phasic solution of phenol and guanidine isothiocyanate, is an improvement to the single-step RNA isolation method. During sample homogenization or lysis, TRIpure Reagent maintains the integrity of the RNA, while disrupting cells and dissolving cell components.
Addition of chloroform followed by centrifugation, separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase. After transfer of the aqueous phase, the RNA is recovered by precipitation with isopropyl alcohol. After removal of the aqueous phase, the DNA and proteins in the sample can be recovered by sequential precipitation. Precipitation with ethanol yields DNA from the interphase, and an additional precipitation with isopropyl alcohol yields proteins from the organic phase. Copurification of the DNA may be useful for normalizing RNA yields from sample to sample.
This technique performs well with small quantities of tissue (50-100 mg) and cells (5 × 106), and large quantities of tissue (≥1 g) and cells (>107), of human, animal, plant, or bacterial origin.
The simplicity of the TRIpure Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total RNA isolated by TRIpure Reagent is free of protein and DNA contamination. It can be used for Northern blot analysis, dot blot hybridization, poly (A)+ selection, in vitro translation, RNase protection assay, and molecular cloning. For use in the polymerase chain reaction (PCR), treatment of the isolated RNA with amplification grade DNase I is recommended when the two primers lie within a single exon.