Separation and purification of nucleic acid from paraffin-containing samples

Abstract

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 ribonucleic acid from a paraffin-containing sample, comprising:
 (a) heating said sample at about 50-85° C. for about 1-60 minutes in the presence of an ionic detergent to produce a paraffin phase and an aqueous phase;   (b) removing said aqueous phase from said paraffin phase; and   (c) adding a protease to said aqueous phase and incubating said aqueous phase at about 25-80° C. for about 5-60 minutes.   
     
     
         2 . The method of  claim 1 , wherein said paraffin-containing sample is a formalin fixed paraffin embedded (FFPE) tissue sample. 
     
     
         3 . The method of  claim 1 , wherein said heating is between about 64-85° C. 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein said heating is for about 1-30 min. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein said ionic detergent is sodium dodecyl sulfate (SDS), or Sarcosine. 
     
     
         9 . The method of  claim 1 , wherein said protease is Proteinase K. 
     
     
         10 . The method of  claim 1 , wherein said incubating is at about 35-70° C. 
     
     
         11 - 12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein said incubating is for about 5-30 min. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , further comprising purifying said ribonucleic acid from said aqueous phase. 
     
     
         16 . The method of  claim 15 , wherein said purifying involves TRIZOL precipitation, guanidinium isothiocyanate, anion exchange chromatography, silica-based purification, ChargeSwitch® purification, or nucleic acid hybridization. 
     
     
         17 . A method for separating ribonucleic acid from a paraffin-containing sample, comprising:
 (a) heating said sample at about 50-85° C. for about 1-60 minutes in the presence of an ionic detergent to produce a paraffin phase and an aqueous phase; and   (b) adding a protease to said aqueous phase and incubating said aqueous phase at about 25-80° C. for about 5-60 minutes.   
     
     
         18 . The method of  claim 17 , further comprising removing said aqueous phase from said paraffin phase. 
     
     
         19 . The method of  claim 18 , wherein said paraffin-containing sample is a formalin fixed paraffin embedded (FFPE) tissue sample. 
     
     
         20 . The method of  claim 18 , wherein said heating is between about 64° C. and about 85° C. 
     
     
         21 - 31 . (canceled) 
     
     
         32 . The method of  claim 18 , further comprising purifying said ribonucleic acid from said aqueous phase. 
     
     
         33 . The method of  claim 32 , wherein said purifying involves TRIZOL precipitation, guanidinium isothiocyanate, anion exchange chromatography, silica-based purification, ChargeSwitch® purification, or nucleic acid hybridization. 
     
     
         34 . A method for separating ribonucleic acid from a paraffin-containing sample, comprising heating said sample to about 50-85° C. for about 1-60 minutes in the presence of an ionic detergent and a protease to produce a paraffin phase and an aqueous phase. 
     
     
         35 . The method of  claim 34 , further comprising removing said aqueous phase from said paraffin phase. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 35 , wherein said heating is at about 64-85° C. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . The method of  claim 35 , wherein said heating is for about 1 min to about 30 min. 
     
     
         41 - 81 . (canceled)