Method and an alggorithm for mrna expression analysis

A method of providing a profile of mRNA molecules present in a sample, the method comprising:

• synthesizing a cDNA strand complementary to each mRNA using the mRNA as template, thereby providing a population of first cDNA strands;
• removing the mRNA;
• synthesizing a second cDNA strand complementary to each first strand, thereby providing a population of double-stranded cDNA molecules;
• digesting the double-stranded cDNA molecules with a Type II or Type IIS restriction enzyme to provide a population of digested double-stranded cDNA molecules, each digested double-stranded cDNA molecule having a cohesive end provided by the restriction enzyme digestion;
• ligating a population of adaptor oligonucleotides to the cohesive end of each of the digested double-stranded cDNA molecules, the adaptor oligonucleotides each comprising an end sequence complementary to a cohesive end and a primer annealing sequence, thereby providing double-stranded template cDNA molecules each comprising a first strand and a second strand wherein the first strand of the double-stranded template cDNA molecules each comprise a 3′ terminal adaptor oligonucleotide and the second strand of the double-stranded template cDNA molecules each comprise a 3′ terminal polyA sequence;
• purifying said double-stranded template cDNA molecules;
• performing polymerase chain reaction amplification on the double-stranded template cDNA molecules having a sequence complementary to a 3′ end of an mRNA using a population of first primers and a population of second primers,
• wherein the first primers each comprise a sequence which anneals to a primer annealing sequence of an adaptor oligonucleotide;
• and
• where the restriction enzyme is a Type II enzyme the first primers each comprise at least one 3′ terminal variable nucleotide and optionally more than one 3′ terminal variable nucleotides wherein the variable nucleotide is, or at a corresponding position within the variable nucleotides each first primer has, a nucleotide selected from A, T, C and G, whereby the population of first primers primes synthesis in the polymerase chain reaction of first strand product DNA molecules each of which is complementary to the first strand of a template cDNA molecule that comprises adjacent to the primer annealing sequence within the first strand of the template cDNA molecule a nucleotide or sequence of nucleotides complementary to the variable nucleotide or nucleotides of a first primer within the population of first primers;
• or
• where the restriction enzyme is a Type IIS enzyme the first primers prime synthesis in the polymerase chain reaction of first strand product DNA molecules each of which is complementary to the first strand of a template cDNA molecule that comprises within the first strand of the template cDNA molecule a sequence of nucleotides complementary to an end sequence of an adaptor oligonucleotide in the population of adaptor oligonucleotides;
• the second primers comprise an oligoT sequence and a 3′ variable portion conforming to the following formula:
• (G/C/A)(X)n wherein X is any nucleotide, n is zero, at least one or more than one;
• whereby the population of second primers primes synthesis in the polymerase chain reaction of second strand product DNA molecules each of which is complementary to the second strand of a template cDNA molecule that comprises adjacent to polyA within the second strand of the template cDNA molecule a nucleotide or nucleotides complementary to the variable portion of a second primer within the population of second primers;
• whereby the polymerase chain reaction amplification provides a population of double-stranded product DNA molecules each of which comprises a first strand product DNA molecule and a second strand product DNA molecule;
• separating double-stranded product DNA molecules on the basis of length;
• and
• detecting said double-stranded product DNA molecules;
• whereby a pattern for the population of mRNA molecules present in the sample is provided by combination of length of said double-stranded product DNA molecules and (i) first primer variable nucleotide or nucleotides, where a Type II restriction enzyme is employed, or (ii) adaptor oligonucleotide end sequence, where a Type IIS restriction enzyme is employed;
• generating an additional pattern for the sample using a second, different Type II or Type IIS restriction enzyme, and comparing the patterns generated using at least two different Type II or Type IIS restriction enzymes in separate experiments with a database of signals determined or predicted for known mRNA's, by:
• (i) listing all mRNA's in the database which may correspond to a double-stranded product DNA in each experiment, forming a list of mRNA molecules possibly present for each experiment, and
• (ii) for each experiment listing mRNA's which definitely do not correspond to a double-stranded product DNA molecule, forming a list of mRNA molecules definitely not present for each experiment, then
• (iii) removing the mRNA molecules definitely not present from the list of mRNA molecules possibly present for each experiment, and
• (iv) generating a list of mRNA molecules possibly present and mRNA molecules definitely not present by combining each list generated for each experiment in (iii);
• thereby providing a profile of mRNA molecules present in the sample.