Ribonuclease resistant RNA preparation and utilization

Abstract

The present invention relates to nuclease resistant nucleic acids in general and ribonuclease resistant RNAs in particular. Methods of making and using such nucleic acids are disclosed.

1 - 71 . (canceled)  
     
     
         72 . A method of detecting and/or quantifying a cellular mRNA in a sample comprising: 
 (a) obtaining a sample;    (b) obtaining a ribonuclease resistant RNA standard comprising a recombinant RNA segment standard encapsidated in a viral coat protein, wherein the recombinant RNA segment standard comprises a region encoding a sequence that can be a standard for detecting and/or quantifying the cellular mRNA;    (c) employing the recombinant RNA segment standard as a standard to detect and/or quantify the cellular mRNA in the sample; and    (d) detecting and/or quantifying the cellular mRNA in the sample.    
     
     
         73 . The method of  claim 72 , wherein the sample is a tissue sample.  
     
     
         74 . The method of  claim 72 , wherein the sample is a blood sample.  
     
     
         75 . The method of  claim 72 , wherein the cellular mRNA encodes a cytokine, a cell cycle gene, or an oncogene.  
     
     
         76 . The method of  claim 75 , wherein the sequence that can be a standard for detecting and/or quantifying the cellular mRNA is further defined as a sequence that can be a standard for detecting and/or quantifying a cytokine.  
     
     
         77 . The method of  claim 76 , wherein the sequence that can be a standard for detecting and/or quantifying the cytokine comprises a modified cytokine sequence.  
     
     
         78 . The method of  claim 75 , wherein the sequence that can be a standard for detecting and/or quantifying the cellular mRNA is further defined as a sequence that can be a standard for detecting and/or quantifying a cell cycle gene.  
     
     
         79 . The method of  claim 78 , wherein the sequence that can be a standard for detecting and/or quantifying the cell cycle gene comprises a modified cell cycle gene sequence.  
     
     
         80 . The method of  claim 75 , wherein the sequence that can be a standard for detecting and/or quantifying the cellular mRNA is further defined as a sequence that can be a standard for detecting and/or quantifying an oncogene.  
     
     
         81 . The method of  claim 80 , wherein the sequence that can be a standard for detecting and/or quantifying the oncogene comprises a modified oncogene sequence.  
     
     
         82 . The method of  claim 72 , further comprising isolating the RNA segment standard from the viral coat protein.  
     
     
         83 . The method of  claim 72 , further comprising isolating sample RNA from the sample.  
     
     
         84 . The method of  claim 83 , further comprising isolating the RNA segment standard from the viral coat protein.  
     
     
         85 . The method of  claim 84 , further comprising admixing the sample and the standard prior to isolating the RNA segment standard so that isolation of the sample RNA and isolation of the RNA segment standard from the viral coat protein are performed in the same isolation procedure.  
     
     
         86 . The method of  claim 72 , further comprising admixing the sample and the standard prior to quantifying the cellular mRNA.  
     
     
         87 . The method of  claim 72 , wherein the standard used to detect and/or quantify the cellular mRNA in the sample is an internal standard.  
     
     
         88 . The method of  claim 72 , wherein the standard used to detect and/or quantify the cellular mRNA in the sample is an external standard.  
     
     
         89 . The method of  claim 72 , wherein the standard used to detect and/or quantify the cellular mRNA in the sample is a positive control standard.  
     
     
         90 . The method of  claim 72 , wherein the method is a detection assay.  
     
     
         91 . The method of  claim 72 , wherein the method is a quantification assay.  
     
     
         92 . The method of  claim 72 , wherein the method is a detection and quantification assay.  
     
     
         93 . The method of  claim 72 , wherein the method further comprises using a polymerase chain reaction procedure.  
     
     
         94 . The method of  claim 93 , wherein the polymerase chain reaction procedure comprises: 
 employing reverse a reverse transcription procedure;    amplifying a DNA to generate a polymerase chain reaction product; and    detecting the polymerase chain reaction product.    
     
     
         95 . The method of  claim 94 , wherein amplifying comprises co-amplification of a polymerase chain reaction product amplified from the sample RNA and a polymerase chain reaction product amplified from the RNA segment standard.  
     
     
         96 . The method of  claim 95 , wherein the co-amplification involves the use of a single primer set for amplification of both the polymerase chain reaction product amplified from the sample RNA and the polymerase chain reaction product amplified from the RNA segment standard in a reverse transcriptase-polymerase chain reaction procedure.  
     
     
         97 . The method of  claim 95 , wherein the method comprises comparing an amount of polymerase chain reaction product amplified from the sample RNA with an amount of polymerase chain reaction product amplified from the RNA segment standard.  
     
     
         98 . The method of  claim 72 , wherein the viral coat protein is a modified coat protein having a different amino acid sequence as compared to a native sequence.  
     
     
         99 . The method of  claim 72 , wherein the viral coat protein comprises bacteriophage viral coat protein.  
     
     
         100 . The method of  claim 99 , wherein the bacteriophage viral coat protein is of an MS2/R17 bacteriophage.