TRI Reagent (r) is used to homogenize or lyse biological samples; the addition of bromochloropropane or chloroform results in the separation of the homogenate into aqueous and organic phases. RNA enters the aqueous phase, DNA enters the interphase, and proteins enter the organic phase. The aqueous phase containing the RNA can be further purified using RNeasy kits (or any other method suitable for your laboratory protocol). The DNA in the interphase can be extracted using standard phenol-chloroform protocols or with commercial spin column kits.
The TRI Reagent protocol is as follows: 1. Thaw on ice. 2. Add 500 μL of TRI Reagent per sample. 3. Mix by inverting several times. 4. Let stand at room temperature for 5 min. 5. Centrifuge at 12000 rpm for 15 min at 4°C. 6. Transfer the upper aqueous phase to a new tube. 7. Add 250 μL of 100% ethanol per sample. 8. Mix by inverting several times. 9. Store at -80°C overnight or longer.
1. Wash the RNeasy Minikit plate with RW1 buffer twice, then wash it with 70% ethanol once. 2. Add 200 μL of RNase-free water to each well of the RNeasy Minikit plate.
The material separates into three phases during centrifugation: the bottom organic phase, a middle phase containing denatured proteins and gDNA, and an upper aqueous phase containing RNA. Alcohol precipitation and rehydration are used to recover the upper aqueous phase and collect the RNA. These procedures remove contaminants that would otherwise inhibit downstream processing or analysis.
Centrifugation is often used in molecular biology labs to separate cellular components for further study or processing. For example, it can be used to isolate DNA from cells or tissues of interest. Centrifugation is also commonly used to purify RNA. The procedure described here is called "phenol-chloroform extraction". Other methods include using acid guanidinium thiocyanate, which dissolves only RNA that has not been degraded by enzymes; however, this method requires additional steps to obtain pure RNA samples free of genomic DNA.
In addition to these methods, newer techniques such as magnetic beads coated with oligonucleotides can be used to capture specific sequences of RNA. This method allows selective enrichment of small amounts of RNA present in a complex mixture.
Finally, high speed centrifugation can be used to rapidly pelletize cells or tissue samples down to a granular state before extracting their RNA. This process, called "dry ice grinding", is useful when working with samples that tend to stick to glass or plasticware during standard isolation procedures.
TRIzol (tm) Reagent preserves RNA integrity by inhibiting RNase activity while disrupting cells and dissolving cell components during sample homogenization. As such, TRIzol (tm) Reagent is a useful tool for isolating high-quality RNA from a wide variety of biological samples.
RNA is very stable when frozen at -20°C or colder. Longer term storage may cause degradation. Refer to the manufacturer's instructions for recommendations on long-term storage.
Once thawed, RNA remains stable for up to 24 hours at room temperature or three days if frozen at -80°C.
For optimal results, isolate RNA as soon as possible after removing it from storage.
The concentration of RNA in TRIzol (tm) Reagent protects it from degradation. The maximum recommended concentration for clinical use is 10 mg/ml. Higher concentrations may be used for RNA extraction from some sources such as blood, but this should be done with caution because higher concentrations may lead to over-amplification of certain genes.
Clinical samples should be treated with care to prevent contamination with DNA. To avoid genomic DNA contamination, all glassware and equipment used for RNA isolation must be DNAse free.