Method and Kit For The Isolation Of Genomic DNA, RNA Proteins and Metabolites From A Single Biological Sample
AssigneesCentre De Recherche Public - Gabriel Lippmann, Universite Du Luxembourg
InventorsPaul Wilmes, Hugo Roume, Karsten Hiller, Thekla Cordes
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Abstract
The invention provides a method and kit for the separation and purification of cellular components including polar and non-polar metabolites, genomic DNA, RNA and proteins from a single biological sample where two steps of lysis of the cells are performed sequentially, before and after a metabolite isolation step. The first lysis step is mechanical and performed in order to be incomplete, whereas the second is chemical or both mechanical and chemical. A sequential isolation of genomic DNA, RNA and proteins is carried out after the second lysis step.
Claims21
-15. (canceled)
A method for the separation and purification of cellular components from a single biological sample, the cellular components comprising polar and non-polar metabolites, genomic DNA, RNA and proteins, wherein the method comprises the following steps:
- a. performing a mechanical lysis and homogenization of the single biological sample such that a part of the cells are lysed, the mechanical lysis being halted when about 30 to 60% of cells have been lysed;
- b. performing a metabolite extraction on the homogenized single biological sample from step (a) by addition of a phase separation solution, homogenization and centrifugation to form an upper phase, an interphase pellet and a lower phase;
- such that polar metabolites are in the upper phase, genomic DNA, RNA and proteins and the remaining cells not lysed by the mechanical lysis are in the interphase pellet, and non-polar metabolites are in the lower phase;
- c. collecting separately the upper phase, the lower phase and the interphase pellet;
- d. adding a lysis solution to the collected interphase pellet to perform a chemical lysis or a combined mechanical and chemical lysis, in order to obtain a lysate;
- e. performing a sequential isolation of genomic DNA, RNA and proteins on the lysate.
The method as claimed in claim 31 wherein the phase separation solution consists of a mixture of methanol and chloroform and water in the proportion of 1 volume of methanol, 1 volume of supernatant and two volumes of chloroform.
Dependencies are inferred from the claim text.