Methods For Determining Biological States By Assessing Amounts Of Target Nucleic Acids In A Sample

Abstract

Described herein are compositions for evaluating nucleic acids, standardized mixture for assessing amounts of at least one target nucleic acid in a sample, and kits comprising the same.

What is claimed is: 
     
         1 . A method for obtaining a numerical index that indicates a biological state, comprising:
 providing two samples corresponding to each of a first biological state and a second biological state;   assessing an amount of each of two nucleic acids in each of said two samples wherein said assessing can enumerate less than about 1,000 molecules of each of said two nucleic acids;   providing said amounts as numerical values wherein said numerical values are directly comparable between a number of samples;   mathematically computing said numerical values corresponding to each of said first and said second biological states; and   determining a mathematical computation that discriminates said first and said second biological states, thereby obtaining said numerical index.   
     
     
         2 . The method as recited in  claim 1 , wherein said two nucleic acids are associated with said first biological state and not with said second biological state. 
     
     
         3 . The method as recited in  claim 2 , wherein one of said two nucleic acids is positively associated with said first biological state and the other of said two nucleic acids is negatively associated with said first biological state. 
     
     
         4 . The method as recited in  claim 3 , wherein said mathematical computation comprises dividing a numerator by a denominator, said numerator corresponding to said nucleic acid positively associated with said first biological state and said denominator corresponding to said nucleic acid negatively associated with said first biological state. 
     
     
         5 . The method as recited in  claim 1 , wherein said first biological state is a disease state and said second biological state is a non-disease state. 
     
     
         6 . The method as recited in  claim 1 , wherein the biological state includes: a disease phenotype; a predisposition to a disease state or a non-disease state; a therapeutic drug response or predisposition to such a response; an adverse drug response or a predisposition to such a response, a resistance to a drug; and/or a predisposition to showing such a resistance. 
     
     
         7 . The method as recited in  claim 1 , wherein the numerical index obtained is a biomarker that correlates with a phenotype of interest to provide personalized medicine. 
     
     
         8 . The method as recited in  claim 1 , wherein the numerical indices are directly comparable between a number of samples. 
     
     
         9 . The method as recited in  claim 8 , wherein the samples obtained from different subjects and/or from different species. 
     
     
         10 . The method as recited in  claim 1 , wherein the numerical indices are directly comparable between a number of samples measured and/or enumerated in different laboratories and/or at different times. 
     
     
         11 . The method as recited in  claim 1 , wherein biological state is an angiogenesis-related condition, an antioxidant-related condition, an apotosis-related condition, a cardiovascular-related condition, a cell cycle-related condition, a cell structure-related condition, a cytokine-related condition, a defense response-related condition, a development-related condition, a diabetes-related condition, a differentiation-related condition, a DNA replication and/or repair-related condition, an endothelial cell-related condition, an folate receptor-related condition, an hormone receptor-related condition, an inflammation-related condition, an intermediary metabolism-related condition, a membrane transport-related condition, an oxidative metabolism-related condition, neurotransmission-related condition, a cancer-related condition, a protein maturation-related condition, a signal transduction-related condition, a stress response-related condition, a tissue structure-related condition, a transcription factor-related condition, a transport-related condition, or a xenobiotic metabolism-related condition. 
     
     
         12 . The method as recited in  claim 1 , wherein said biological state corresponds to a normal expression level of a first gene. 
     
     
         13 . The method as recited in  claim 1 , wherein more than two biological states are distinguished. 
     
     
         14 . The method as recited in  claim 1 , wherein the samples are provided from a range of biological states, including corresponding to different stages of disease progression, and/or different stages of cancer. 
     
     
         15 . The method as recited in  claim 14 , wherein the different stages of cancer include a non-cancerous cell vs. a non-metastasizing cancerous cell vs. a metastasizing cell from a given patient at various times over the disease course. 
     
     
         16 . The method as recited in  claim 15 , wherein the cancer cells of various types of cancer include a bladder cancer, a bone cancer, a brain tumor, a breast cancer, a colon cancer, an endocrine system cancer, a gastrointestinal cancer, a gynecological cancer, a head and neck cancer, a leukemia, a lung cancer, a lymphoma, a metastases, a myeloma, neoplastic tissue, a pediatric cancer, a penile cancer, a prostate cancer, a sarcoma, a skin cancer, a testicular cancer, a thyroid cancer, and/or a urinary tract cancer. 
     
     
         17 . The method as recited in  claim 15 , wherein a non-cancerous includes a cell of hematoma and/or scar tissue, as well as morphologically normal parenchyma from non-cancer patients and/or non-cancer patients related or not related to a cancer patient. 
     
     
         18 . The method as recited in  claim 1 , wherein non-cancerous cells include morphologically normal parenchyma from cancer patients; from a site close to the site of the cancer in the same tissue and/or same organ; from a site further away from the site of the cancer; in a different tissue and/or organ in the same organ-system, or from a site still further away; and/or in a different organ and/or a different organ-system. 
     
     
         19 . The method as recited in  claim 1 , further comprising a database where measured amounts of the biological states are mathematically combined to provide numerical indices that allow for direct inter-experiment comparison. 
     
     
         20 . The method as recited in  claim 19 , wherein the database establishes a continuously expanding multiplex experiment that includes data from an ever-expanding number of nucleic acids, samples and/or specimens that are used to calculate numerical indices and that can be and compared directly to other data within the database. 
     
     
         21 . The method as recited in  claim 1 , wherein any measured nucleic acid or combination of nucleic acids, including all measured nucleic acids, can be used as the reference gene and data calculated using a first reference nucleic acid can be re-calculated relative to that of another reference nucleic acid. 
     
     
         22 . The method as recited in  claim 1 , wherein, in the case of numerical indices, the difference in value obtained after converting from one reference nucleic acid to another depends on how many numerical values are in the numerator and how many are in the denominator. 
     
     
         23 . The method as recited in  claim 1 , wherein each numerical value in a numerical index is converted to the new reference in calculating the index. 
     
     
         24 . The method as recited in  claim 1 , wherein when there are equal numbers of numerical values in the numerator and denominator, conversion to a new reference has no effect on the relative numerical index between samples and/or specimen. 
     
     
         25 . The method as recited in  claim 1 , wherein when there are non-equal numbers of numerical values in the numerator and denominator, the relative numerical index between samples and/or specimen changes in accordance with a difference in relative numerical values for the reference nucleic acids between the samples and/or specimen. 
     
     
         26 . A method of comparing different nucleic acids amplified in separate vessels to be directly compared, comprising
 using a standardized mixture, wherein the use of a standardized mixture allows the concentration of internal standard for a nucleic acid relative to others to remain fixed across different measurements.   
     
     
         27 . The method according to  claim 26 , wherein one nucleic acid and its competitive template are co-amplified in one vessel, while another nucleic acid and its competitive template are co-amplified in a different vessel. 
     
     
         28 . The method according to  claim 26 , wherein use of a common standardized mixture allows direct comparisons to be made among samples, and the different samples may be amplified at different times, on different days; in the same or different experiments in the same laboratory; and/or in different experiments in different laboratories. 
     
     
         29 . The method according to  claim 26 , wherein measurements are made using the same standardized mixture and dilution of internal standard competitive templates. 
     
     
         30 . The method according to  claim 26 , wherein measurements obtained using various quantifying approaches are directly comparable where a common standardized mixture is used. 
     
     
         31 . The method according to  claim 26 , wherein the use of the standardized mixtures is used for measuring nucleic acids in real-time RT-PCR. 
     
     
         32 . The method according to  claim 31 , wherein real-time RT-PCR analyses are used to obtain a ratio of amplified product of a nucleic acid to amplified product of a competitive template for the nucleic acid comprises a use of real-time RT-PCR analyses.