Separation and purification of nucleic acid from paraffin-containing samples
Disclosed are rapid and reproducible methods for separating and purifying nucleic acids from paraffin-containing tissue samples. The disclosed methods involve a melting step, where paraffin-containing tissue samples are heated in the presence of a detergent, causing the paraffin to melt and tissue sample cells to lyse. From the resulting two-phase mixture (i.e., a paraffin phase and an aqueous phase), the aqueous phase is collected for purification of nucleic acid. Protease(s) can be used at one or more points in the separation process to facilitate tissue cell lysis and/or degrade proteins that could degrade nucleic acid or interfere with subsequent genetic manipulation or analysis.
1. A method for separating micro ribonucleic acid from a paraffin-containing sample comprising:
(a) heating the sample at a temperature of from 50° C. to 85° C. for from 1 to 60 minutes in the presence of an ionic detergent and a protease,
(b) remove the aqueous phase from the sample,
(c) add ethanol to the aqueous phase to a final concentration of 35% and apply to a first silica column,
(d) collect the eluate from the silica column of step (c),
(e) adjust the ethanol concentration of the eluate of step (d) to 75% and apply to a second silica column and;
(f) elute the micro ribonucleic acid from the second silica column using water.
2. The method of claim 1 , wherein the ionic detergent is sodium dodecyl sulfate.
3. The method of claim 1 , wherein the protease is Proteinase K.
4. The method of claim 1 , wherein the sample is heated for 10 minutes at 75° C.
5. The method of claim 1 , wherein the micro ribonucleic acid is from 15 to 75 bases long.