Simultaneous extraction of DNA and RNA from FFPE tissues
View Patent ↗The present provides methods and kits for extracting DNA and RNA from FFPE tissues and samples.
1. A method of recovering both RNA and DNA from a biological sample comprising contacting the sample with a monophasic solution comprising phenol and guanidine isothiocyanate,
obtaining RNA from the resultant upper phase,
contacting the resultant lower phase with an amount of ethanol and centrifuging for a time sufficient to create a pellet,
contacting the pellet with an amount of a first buffer solution and proteinase K,
purifying DNA from the pellet by contacting with an amount of a second buffer and an amount of ethanol,
binding, washing and eluting the purified DNA so as to obtain the DNA,
thereby recovering both RNA and DNA from the same biological sample.
2. The method of claim 1 , wherein the monophasic solution also comprises ammonium thiocyanate.
3. The method of claim 1 , wherein the phenol is 30-60% of the monophasic solution and the guanidine isothiocyanate is 15-40% of the monophasic solution.
4. The method of claim 1 , wherein the monophasic solution comprises ammonium thiocyanate at 7-13% of the solution.
5. The method of claim 1 , wherein the biological sample comprises a formalin-fixed and paraffin-embedded biological sample.
6. The method of claim 1 , wherein the biological sample comprises serum, plasma, blood, urine, tears, saliva, or semen.
7. The method of claim 1 , wherein the biological sample comprises a purified exosome.
8. The method of claim 1 , wherein obtaining RNA from the resultant upper phase comprises contacting the upper phase with isopropanol, sodium acetate and linear acrylamide then centrifuging with ethanol a sufficient amount of times to obtain a pellet, air drying the pellet, contacting with buffer and centrifuging a further amount of time so as to obtain the RNA.
9. The method of claim 1 , wherein the biological sample is formalin-fixed.
10. The method of claim 1 , wherein the biological sample is paraffin-embedded.
11. The method of claim 1 , further comprising, after contacting the resultant lower phase with an amount of ethanol and centrifuging at a temperature below 10° C. for a time sufficient to create a pellet, air drying the pellet and centrifuging it again in an amount of 80% ethanol and then air-drying the pellet, prior to contacting the pellet with an amount of a first buffer solution and proteinase K.
12. The method of claim 1 , wherein the pellet is contacted with the first buffer solution and proteinase K for at least a 20 hour period, and then washed and contacted with a second amount of a first buffer solution and proteinase K for at least a second 20 hour period.
13. The method of claim 1 , wherein the pellet is contacted with the first buffer solution and proteinase K for at least a 24 hour period, and then washed and contacted with a second amount of a first buffer solution and proteinase K for at least a second 24 hour period.
14. The method of claim 1 , wherein the sample comprises genomic DNA.
15. The method of claim 1 , wherein the sample comprises microRNA.
16. The method of claim 1 , further comprising digesting the biological sample prior to contacting it with the monophasic solution.
17. The method of claim 1 , wherein the contacting the pellet with an amount of a first buffer solution and proteinase K is performed for a time sufficient to effect digestion of the majority of cross-linked proteins in the pellet.
18. The method of claim 1 , wherein, in contacting the resultant lower phase with an amount of ethanol, the centrifuging occurs at a temperature below 10° C. for a time sufficient to create a pellet.
19. A kit for performing the method of claim 1 comprising
an amount of powdered Proteinase K;
an amount of a chaotropic reagent for separation of RNA and DNA fractions;
one or more binding columns to recover the DNA;
optionally, if the RNA is not to be precipitated, a binding column to recover the RNA;
optionally, if the RNA is to be precipitated, an amount of linear acrylamide for performing precipitation of RNA;
one or more binding buffers for the binding columns;
one or more wash buffers for the binding columns;
RNAse/DNase-free water for elution of DNA from binding columns;
and instructions for use.