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Epicentre Forum 3 (3)
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Recovery and purification of RNA from in vitro transcription
reactions can be tedious, time-consuming and result in significant product
loss. For example, one of the more common methods involves digestion
of the DNA template using RNase-free DNase I, followed by gel filtration
to remove unincorporated nucleotides, and then phenol extraction to remove
proteins and ethanol precipitation to concentrate the RNA. A second standard
method is purification of the RNA by polyacrylamide gel electrophoresis,
then extraction of the RNA from the gel by electroelution.
Table 1 presents an easier and faster method. Ammonium acetate selectively
precipitates only RNA, leaving DNA, proteins, and unincorporated nucleotides
in the supernatant. Recovery of RNA transcripts longer than 100 bases
approaches 100%.
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Table 1. Selective RNA Precipitation with Ammonium
Acetate
- Add ammonium acetate solution to a final concentration of 2.5
M (e.g., add one volume of 5 M ammonium acetate and mix). Note:
If the transcription reaction volume is less than 100 µl,
bring the reaction to a 100µl final volume with RNase-free
H2O prior to adding ammonium acetate.
- Chill the mixture on ice for 15 minutes.
- Centrifuge in a microcentrifuge at high speed (approx. 10,000
x g) for 15 minutes.
- Remove the supernatant and wash the pellet with 70% ethanol
to remove salts.
- Resuspend the RNA pellet in the desired volume of RNase-free
H2O or buffer.
- Quantitate the RNA by an appropriate method.
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Note: Since ammonium acetate decomposes by loss of ammonia,
solutions should be prepared only from the pure salt which has been kept
cool in a closed container. Sterilize only by filtration; do not autoclave.
Store solutions at +4°C.
*Reprinted with minor modifications from Epicentre Forum 1 (2).
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