TISSUE HOMOGENIZATION BUFFER FOR EFFICIENT QUANTITATION OF A THERAPEUTIC OLIGONUCLEOTIDE IN MULTIPLE TISSUES
Embodiments of the present invention provide for an optimized tissue homogenization buffer composition and a method of using an optimized tissue homogenization buffer for efficient quantitation of a therapeutic oligonucleotide in multiple tissues. The exemplary method comprising: mixing the tissue with a homogenization buffer composition to create a tissue homogenate; adding the tissue homogenate to a quantity of oligonucleotide-free plasma to create a tissue homogenate solution; adding a quantity of a reference standard oligonucleotide to the tissue homogenate solution to create a homogenate/plasma/standard solution; adding a quantity of a phenol/chloroform/isoamyl alcohol to the homogenate/plasma/standard solution; employing centrifugal force to separate a supernatant from the homogenate/plasma/standard solution; and analyzing the supernatant for a concentration of the oligonucleotide.
1 . A method for quantitation of an oligonucleotide within a tissue, the method comprising:
mixing the tissue with a homogenization buffer composition to create a tissue homogenate, wherein the homogenization buffer composition comprises ammonium acetate in solution in water;
adding the tissue homogenate to a quantity of oligonucleotide-free plasma to create a tissue homogenate solution;
adding a quantity of a reference standard oligonucleotide to the tissue homogenate solution to create a homogenate/plasma/standard solution;
adding a quantity of a phenol/chloroform/isoamyl alcohol to the homogenate/plasma/standard solution;
employing centrifugal force to separate a supernatant from the homogenate/plasma/standard solution; and
analyzing the supernatant for a concentration of the oligonucleotide, wherein the oligonucleotide is a toll-like receptor 9 agonist oligonucleotide having the structure: 5′-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3′ (SEQ ID NO: 1).
2 . The method of claim 1 , wherein a concentration of the ammonium acetate within the homogenization buffer composition is about 5 mM to 20 mM.
3 . The method of claim 2 , wherein the concentration of the ammonium acetate within the homogenization buffer composition is 10 mM.
4 . The method of claim 1 , wherein the pH of the homogenization buffer composition is between 3.0 to 6.0 pH.
5 . The method of claim 4 , wherein the pH of the homogenization buffer composition is 4.5 pH.
6 . The method of claim 1 , wherein the homogenization buffer composition is in a concentration ranging from 4 mL of the homogenization buffer composition per 1 gram of the tissue to 200 mL of the homogenization buffer composition per 1 gram of the tissue.
7 . The method of claim 6 , wherein the homogenization buffer composition is in a concentration ranging from 20 mL of the homogenization buffer composition per 1 gram of the tissue to 100 mL of the homogenization buffer composition per 1 gram of the tissue.
8 . The method of claim 7 , wherein the homogenization buffer composition is in a concentration ranging from 20 mL of the homogenization buffer composition per 1 gram of the tissue to 50 mL of the homogenization buffer composition per 1 gram of the tissue.
9 . The method of claim 8 , wherein the homogenization buffer composition is in a concentration of 50 mL of the homogenization buffer composition per 1 gram of the tissue.
10 . The method of claim 1 , wherein the homogenization buffer composition further comprises a quantity of proteinase K.
11 . The method of claim 10 , wherein the quantity of proteinase K is from 50 μg to 200 μg for every 1 mL of the homogenization buffer composition.
12 . The method of claim 11 , wherein the quantity of proteinase K is 50 μg for every 1 mL of the homogenization buffer composition.
13 . The method of claim 1 , wherein the tissue homogenate solution comprises oligonucleotide-free plasma and tissue homogenate at a 50/50 volume ratio.
14 . The method of claim 1 , wherein the homogenate/plasma/standard solution comprises 200 μL of tissue homogenate, 200 μL of oligonucleotide-free plasma, and 20.0 μL of reference standard oligonucleotide.
15 . The method of claim 1 , wherein the quantity of the phenol/chloroform/isoamyl alcohol is 100 μL.
16 . The method of claim 1 , wherein the phenol/chloroform/isoamyl alcohol is 25/24/1 phenol/chloroform/isoamyl alcohol v/v.
17 . The method of claim 1 , wherein the supernatant is analyzed for the concentration of the oligonucleotide by high performance liquid chromatography tandem mass spectroscopy (HPLC-MS/MS).
18 . The method of claim 1 , wherein the tissue is any one of liver, lung, brain, spleen, thymus, and lymphoid tissue.
19 . The method of claim 18 , wherein the tissue does not include any cancerous tumors.
20 . A homogenization buffer composition for quantitation of an oligonucleotide within a tissue, the homogenization buffer composition comprising:
ammonium acetate in solution in water, wherein a concentration of the ammonium acetate within the homogenization buffer composition is about 5 mM to 20 mM; and
a quantity of proteinase K, wherein the quantity of proteinase K is from 50 μg to 200 μg for every 1 mL of the homogenization buffer composition;
wherein:
the pH of the homogenization buffer composition is between 3.0 to 6.0 pH; and
the homogenization buffer composition is in a concentration ranging from 4 mL of the homogenization buffer composition per 1 gram of the tissue to 200 mL of the homogenization buffer composition per 1 gram of the tissue.