Method for simultaneous identification of differentially expressed mRNAs and measurement of relative concentrations

Claim 1

A method for simultaneous sequence-specific identification of mRNAs in a mRNA population comprising the steps of: (a) isolating a mRNA population; (b) preparing double-stranded cDNAs from the mRNA population using a mixture of 12 anchor primers with the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 2), wherein V is a deoxyribonucleotide selected from the group consisting of A, C, and G; and N is a deoxyribonucleotide selected from the group consisting of A, C, G, and T, the mixture including anchor primers containing all possibilities for V and N, to produce a cDNA sample; (c) cleaving the cDNA sample with two restriction endonucleases, a first restriction endonuclease MspI and a second restriction endonuclease NotI; (d) inserting the cDNA sample cleaved with the first and second restriction endonucleases into a vector, the cleaved cDNA being inserted in an orientation that is antisense with respect to a T3 promoter within the vector, the vector being the plasmid pBC SK + cleaved with ClaI and NotI; (e) transforming Escherichia coli with the vector into which the cleaved cDNA has been inserted to produce cloned inserts; (f) generating linearized fragments of the cloned inserts by digestion with at least one restriction endonuclease that is different from the first and second restriction endonucleases; (g) generating a cRNA preparation of antisense cRNA transcripts by incubation of the linearized fragments with a T3 RNA polymerase capable of initiating transcription from the T3-specific promoter; (h) dividing the cRNA preparation into sixteen subpools and transcribing first-strand cDNA from each subpool, using a thermostable reverse transcriptase, and one of the sixteen primers A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO: 3), wherein N is one of the four deoxyribonucleotides A, C, G, or T, and a different primer is used in each of the subpool (i) using the product of transcription in each of the sixteen subpools as a template for a polymerase chain reaction with the 3'-primer G-A-A-C-A-A-A-A-G-C-T-G-G-A-G-C-T-C-C-A-C-C-G-C (SEQ ID NO: 4), and a 5'-primer selected from the group consisting of: (1) the primer from which first-strand cDNA was made for that subpool; (2) the primer from which the first-strand cDNA was made for that subpool extended at its 3'-terminus by an additional residue -N, where N can be any of A, C, G, or T; and (3) the primer used for the synthesis of first-strand cDNA for that subpool extended at its 3'-terminus by two additional residues -N-N, wherein N can be any of A, C, G, or T, to produce polymerase chain reaction amplified fragments; and (j) resolving the polymerase chain reaction amplified fragments by electrophoresis to display bands representing the 3'-ends of mRNAs present in the sample.

Claim 2

A method for simultaneous sequence-specific identification of mRNAs in a mRNA population comprising the steps of: (a) isolating a mRNA population; (b) preparing double-stranded cDNAs from the mRNA population using a mixture of 12 anchor primers with the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 2), wherein V is a deoxyribonucleotide selected from the group consisting of A, C, and G; and N is a deoxyribonucleotide selected from the group consisting of A, C, G, and T, the mixture including anchor primers containing all possibilities for V and N, to produce a cDNA sample; (c) cleaving the cDNA sample with two restriction endonucleases, a first restriction endonuclease MspI and a second restriction endonuclease NotI; (d) inserting the cDNA sample cleaved with the first and second restriction endonucleases into a vector, the cleaved cDNA being inserted in an orientation that is antisense with respect to a T3 promoter within the vector, the vector being the plasmid pBC SK + cleaved with ClaI and NotI; (e) transforming Escherichia coli with the vector into which the cleaved cDNA has been inserted to produce cloned inserts; (f) generating linearized fragments of the cloned inserts by digestion with at least one restriction endonuclease that is different from the first and second restriction endonucleases; (g) generating a cRNA preparation of antisense cRNA transcripts by incubation of the linearized fragments with a T3 RNA polymerase capable of initiating transcription from the T3-specific promoter; (h) dividing the cRNA preparation into sixteen subpools and transcribing first-strand cDNA from each subpool, using a thermostable reverse transcriptase, and one of the sixteen primers A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO: 3), wherein N is one of the four deoxyribonucleotides A, C, G, or T, and a different primer is used in each of the subpool (i) using the product of transcription in each of the sixteen subpools as a template for a polymerase chain reaction with the 3'-primer G-A-A-C-A-A-A-A-G-C-T-G-G-A-G-C-T-C-C-A-C-C-G-C (SEQ ID NO: 4), and a 5'-primer selected from the group consisting of: (1) the primer from which first-strand cDNA was made for that subpool; (2) the primer from which the first-strand cDNA was made for that subpool extended at its 3'-terminus by an additional residue -N, where N can be any of A, C, G, or T; and (3) the primer used for the synthesis of first-strand cDNA for that subpool extended at its 3'-terminus by two additional residues -N-N, wherein N can be any of A, C, G, or T, to produce polymerase chain reaction amplified fragments; and (j) resolving the polymerase chain reaction amplified fragments by electrophoresis to display bands representing the 3'-ends of mRNAs present in the sample.