Methods for characterizing mRNA molecules
AssigneeBiofrontera Pharmaceuticals GmbH
InventorsRalf Hoffmann, Hermann Lübbert, Stefan Zwilling
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Abstract
The invention relates to methods for the qualitative and quantitative determination of differentially expressed mRNA molecules. Said methods are used especially to determine all possible mRNA molecules present in a cell or a tissue, and to compare them with other cells or tissues, with other conditions (stages of disease or development), or with stages of treatment for these conditions. The method provided for in the invention therefore makes it possible, for example, to establish a comprehensive map of the different mRNA molecules present in a defined mRNA population and subsequently to use the preferably digital information obtained in this way in data base analyses.
Claims27
A method for identifying and characterizing mRNA molecules, comprising the steps of:
- a) isolating and purifying poly-A mRNA from tissue samples;
- b) synthesizing double-stranded cDNA from the poly-A mRNA, wherein a first cDNA strand is synthesized using reverse transcriptase using an anchored oligo-T nucleotide having a 3′ extension of two bases, wherein a first base is A, C or G and a second base is A, C, G or T, and having a 5′ extension of 5-15 bases, wherein the 5′ extension comprises a recognition site for a second restriction endonuclease with cleavage characteristics {fraction (16/14)} nucleotides downstream of the recognition sequence, and a second cDNA strand is synthesized using the first cDNA strand as a template to form the double-stranded cDNA;
- c) truncating the double-stranded cDNA with a first restriction endonuclease, thereby forming truncated cDNA fragments;
- d) hybridizing and ligating a first set of adaptor molecules to ends of the truncated cDNA fragments;
- e) purifying 3′ ends of the truncated cDNA fragments comprising 3′ poly-da nucleotides;
- f) removing 3′ poly-dA nucleotides from the ends purified ends of the purified truncated cDNA fragments with the second restriction endonuclease;
- g) hybridizing and ligating a second set of adaptor molecules to the truncated cDNA with removed 3′ poly-dA nucleotides;
- h) amplifying the truncated cDNA fragments with removed 3′ poly-da nucleotides with a polymerase chain reaction, thereby forming amplification products, wherein 5′ primers having two or three permutations at the 3′ end and containing one or two artificial mismatches to a complementary adaptor sequence are used in the polymerase chain reaction;
- i) fractionating the amplification products according to their length;
- and,
- j) analyzing the amplification products.
A method for identifying and characterizing mRNA molecules, comprising the steps of:
- a) isolating and purifying poly-A mRNA from tissue samples;
- b) synthesizing double-stranded cDNA from the poly-A mRNA, wherein a first cDNA strand is synthesized using reverse transcriptase using an anchored oligo-T nucleotide having a 3′ extension of two bases, wherein a first base is A, C or G and a second base is A, C, G or T, and having a 5′ extension of 5-15 bases, wherein the 5′ extension comprises a recognition site for a second restriction endonuclease with cleavage characteristics {fraction (16/14)} nucleotides downstream of the recognition sequence, and a second cDNA strand is synthesized using the first cDNA strand as a template to form the double-stranded cDNA;
- c) truncating the double-stranded cDNA with a first restriction endonuclease, thereby forming truncated cDNA fragments with overhangs;
- d) adding deoxyribonucleotides to the overhangs of the truncated cDNA fragments with Klenow DNA polymerase followed by blunt ligation of a first set of adaptor molecules;
- e) purifying the truncated cDNA fragments comprising 3′ poly-dA nucleotides;
- f) removing 3′ poly-dA nucleotides from the ends purified ends of the purified truncated cDNA fragments with the second restriction endonuclease;
- g) hybridizing and ligating a second set of adaptor molecules to the truncated cDNA with removed 3′ poly-dA nucleotides;
- h) amplifying the truncated cDNA fragments with removed 3′ poly-dA nucleotides with a polymerase chain reaction, thereby forming amplification products;
- i) fractionating the amplification products according to their length;
- and,
- j) analyzing the amplification products.
Dependencies are inferred from the claim text.