Microassay for serial analysis of gene expression and applications thereof

Abstract

A method of obtaining a library of tags able to define a specific state of a biological sample, such as a tissue or a cell culture. The present method provides an important advantage over other methods used to analyze gene expression in that libraries may be generated from tiny amounts of cells, e.g., from 30,000-100,000 cells.

1 . A method of obtaining a library of tags capable of defining a specific state of a biological sample, comprising: 
 (1) extracting mRNA from the biological sample by contacting the biological sample with an oligo(dT) covalently bound to paramagnetic beads,    (2) generating a double-strand cDNA library from the extracted mRNA according to a process comprising: 
 (a) synthesizing the 1 st  strand of the cDNA by reverse transcription of the mRNA template into a 1 st  complementary single-strand cDNA, using a reverse transcriptase lacking Rnase H activity, and  
 (b) synthesising the 2 nd  strand of the cDNA by nick translation of the mRNA in the mRNA-cDNA hybrid form by an  E. coli  DNA polymerase,  
   (3) cleaving the cDNAs with an anchoring enzyme, wherein the anchoring enzyme is a restriction endonuclease having a 4-bp recognition site,    (4) separating the cleaved cDNAs in two aliquots,    (5) linking the cDNA contained in each of the two aliquots with two different oligonucleotide linkers comprising a type IIS recognition site,    (6) digesting the products obtained in (5) with a type IIS restriction enzyme, to obtain two different tags,    (7) blunt-ending the tags with a DNA polymerase, and mixing the tags ligated with the different linkers,    (8) ligating the tags obtained in (7) with a DNA ligase, to form ditags, and    (9) determining the nucleotide sequence of at least one tag of the ditags, to detect gene expression.    
     
     
         2 . The method of  claim 1 , wherein the biological sample is a tissue sample or a cell culture.  
     
     
         3 . The method of  claim 1 , wherein the DNA ligase is a T7 DNA polymerase or Vent polymerase.  
     
     
         4 . The method of  claim 1 , wherein the oligo(dT) is an oligo(dT) 25 .  
     
     
         5 . The method of  claim 1 , wherein in (1), after mRNA binding to beads, different washes are performed in washing buffer supplemented with glycogen.  
     
     
         6 . The method of  claim 1 , wherein the two different oligonucleotide linkers further comprise amplification primer hybridization sequences, the method further comprises amplifying the ditags.  
     
     
         7 . The method of  claim 1 , wherein (9) comprises producing and cloning concatemers of the ditags.  
     
     
         8 . The method of  claim 7 , wherein the concatemers of the ditags have more than 300 bp.  
     
     
         9 . The method of  claim 1 , wherein the biological sample of (1) comprises ≦5.10 6  cells, corresponding to at most 50 μg of total RNA or 1 jug of poly(A) RNA.  
     
     
         10 . The method of  claim 1 , wherein the tissue sample is from nephron segments and contains about 15,000 to 45,000 cells, corresponding to 0.15-0.45 μg of total RNA.  
     
     
         11 . A method of obtaining a library of tags able to define a specific state of a biological sample, comprising: 
 (1) extracting mRNA from the biological sample by contacting the biological sample with an oligo(dT) covalently bound to paramagnetic beads,    (2) generating a double-strand cDNA library from the extracted mRNA according to a process comprising: 
 (a) synthesizing the 1 st  strand of the cDNA by reverse transcription of the mRNA template into a 1 st  complementary single-strand cDNA, using a reverse transcriptase lacking Rnase H activity, and  
 (b) synthesising the 2 nd  strand of the cDNA by nick translation of the mRNA in the mRNA-cDNA hybrid form by an  E coli  DNA polymerase,  
   (3) cleaving the cDNAs with the restriction endonuclease Sau3A I as an anchoring enzyme,    (4) separating the cleaved cDNAs in two aliquots,    (5) ligating the cDNA contained in each of the two aliquots via the Sau3A I restriction site to a linker consisting of one double-strand cDNA molecule having one of the following formulas:                                      GATCGTCCC-X 1  or GATCGTCCC-X 2 ,                                 wherein X 1  and X 2 , which comprise 30-37 nucleotides and are different, include a 20-25 bp PCR priming site having a Tm of 55° C.-65° C., and    wherein GATCGTCCC correspond to a Sau3A I restriction site joined to a BsmF I restriction site,    (6) digesting the products from (5) with the tagging enzyme BsmF I and releasing linkers with anchored short piece of cDNA corresponding to a transcript-specific tag, the digestion generating BsmF I tags specific of the initial mRNA,    (7) blunt-ending the BsmF I tags with a DNA polymerase and mixing the tags ligated with the different linkers,    (8) ligating the tags obtained in (7) to form ditags with a DNA ligase,    (9) amplifying the ditags obtained in (8) with primers comprising 20-25 bp and having a Tm of 55°-65° C.,    (10) isolating the ditags having between 20 and 28 bp from the amplification products obtained in (9) by digesting the amplification products with the anchoring enzyme Sau3A I and separating the digested products by gel electrophoresis,    (11) ligating the ditags obtained in (10) to form concatemers, purifying the concatemers, and separating the concatemers having more than 300 bp,    (12) cloning and sequencing the concatemers, and    (13) analyzing the different obtained tags.    
     
     
         12 . The method of  claim 1 , wherein the biological sample is a tissue sample or a cell culture.  
     
     
         13 . The method of  claim 1 , wherein the DNA ligase is a T7 DNA polymerase or Vent polymerase.  
     
     
         14 . The method of  claim 1 , wherein the oligo(dT) is an oligo(dT) 25 .  
     
     
         15 . The method of  claim 1 , wherein in (2), the synthesis of the 1 st  strand of the cDNA is performed with Moloney Murine Leukaemia Virus reverse transcriptase (M-MLV RT), and oligo(dT) 25  as primers.  
     
     
         16 . The method of  claim 1 , wherein the linkers of (5) are hybrid DNA molecules composed linkers 1A and 1B or from linkers 2A and 2B, wherein the linkers have the following formulas:  
       
         
           
                 
                 
               
                     
                 
                   1A: 
                     
                 
                   5′-TTTTGCCAGGTCACTCAAGTCGGTCATTCATGTCAGCACAGGGAC- 
                 
                 
                 
               
                   3′, 
                     
                 
                     
                 
                 
                 
               
                   1B: 
                     
                 
                   5′-GATCGTCCCTGTGCTGACATGAATGACCGACTTGAGTGACCTGGCA- 
                 
                 
                 
               
                   3′, 
                     
                 
                     
                 
                 
                 
               
                   2A: 
                     
                 
                   5′-TTTTTGCTCAGGCTCAAGGCTCGTCTAATCACAGTCGGAAGGGAC- 
                 
                 
                 
               
                   3′, or 
                     
                 
                     
                 
                 
                 
               
                   2B: 
                     
                 
                   5′-GATCGTCCCTTCCGACTGTGATTAGACGAGCCTTGAGCCTGAGCAA- 
                 
                 
                 
               
                   3′. 
                     
                 
                     
                 
             
                
                
                
               
            
             
                
                
               
            
             
                
                
               
            
             
                
                
               
            
             
                
                
               
            
             
                
                
               
            
             
                
                
               
            
             
                
                
               
            
           
         
       
     
     
         17 . The method of  claim 1 , wherein the amount of each linker in (5) is at most of 8-10 pmol and comprised between 0.5 pmol and 8 pmol for initial amounts of respectively 10-40 ng of mRNAs and 5 keg of mRNAs.  
     
     
         18 . The method of  claim 11 , wherein the primers of (9) have the following formulas:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-GCCAGGTCACTCAAGTCGGTCATT-3′, and 
                     
                 
                     
                     
                 
                     
                   5′-TGCTCAGGCTCAAGGCTCGTCTA-3′. 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         19 . The method of  claim 11 , wherein the biological sample of (1) comprises ≦5.10 6  cells, corresponding to at most 50 μg of total RNA or 1 μg of poly(A) RNA.  
     
     
         20 . The method of  claim 1 , wherein the tissue sample is from nephron segments and contains about 15,000 to 45,000 cells, corresponding to 0.15-0.45 μg of total RNA.  
     
     
         21 . The method of  claim 11 , wherein in (1), after mRNA binding to beads, different washes are performed in washing buffer supplemented with glycogen.  
     
     
         22 . A method of determination of a gene expression profile of a biological sample, comprising: 
 performing the method according to  claim 1 , and    translating cDNA tag abundance in gene expression profile.    
     
     
         23 . A method of determination of a gene expression profile of a biological sample, comprising: 
 performing the method according to  claim 11 , and    translating cDNA tag abundance in gene expression profile.    
     
     
         24 . The method of  claim 21 , wherein the gene expression profile obtained in mouse outer medullary collecting duct (OMCD) and in mouse medullary thick ascending limb (MTAL) is as specified in Table I.  
     
     
         25 . The method of  claim 23 , wherein the gene expression profile obtained in mouse outer medullary collecting duct (OMCD) and in mouse medullary thick ascending limb (MTAL) is as specified in Table I.  
     
     
         26 . A kit useful for detection of a gene expression profile, comprising: 
 (a) at least one container which contains an oligonucleotide linker consisting of one double-strand cDNA molecule having one of the following formulas:                                      GATCGTCCC-X 1  or GATCGTCCC-X 2 ,                                 wherein X 1  and X 2 , which comprise 30-37 nucleotides and are different, include a 20-25 bp PCR priming site having a Tm of 55° C.-65° C., and    wherein GATCGTCCC correspond to a Sau3A I restriction site joined to a BsmF I restriction site, and    (b) at least one container which contains primers comprising 20-25 bp and having a Tm of 55° 65° C.    
     
     
         27 . The kit of  claim 26 , further comprising at least one buffer suitable cDNA synthesis, restriction enzyme digestion, DNA ligation, or DNA amplification  
     
     
         28 . The kit of  claim 26 , comprising: 
 at least one container containing a hybrid DNA molecule composed linkers 1A and 1B or linkers 2A and 2B, wherein the linkers have formula:                          1A: 5′-TTTTGCCAGGTCACTCAAGTCGGTCATTCATGTCAGCACAGGGAC-3′,               1B: 5′-GATCGTCCCTGTGCTGACATGAATGACCGACTTGAGTGACCTGGCA-3′,           2A: 5′-TTTTTGCTCAGGCTCAAGGCTCGTCTAATCACAGTCGGAAGGGAC-3′, and           2B: 5′-GATCGTCCCTTCCGACTGTGATTAGACGAGCCTTGAGCCTGAGCAA-3′, and           at least one Container containing the following primers:     5′-GCCAGGTCACTCAAGTCGGTCATT-3′, and           5′-TGCTCAGGCTCAAGGCTCGTCTA-3′.                                    
     
     
         29 . The kit of  claim 28 , comprising a container containing a hybrid DNA molecule composed linkers 1A and 1B and another container containing a hybrid DNA molecule composed linkers 2A and 2B.  
     
     
         30 . The kit of  claim 28 , comprising the primers in different containers.