Referenced amplification of small quantities of RNA

A method of determining the amount of test RNA in a test sample, comprising:

• a) admixing:
• i) a reference sample, wherein the reference sample comprises a known amount of a reference nucleic acid, wherein the reference nucleic acid is labeled with a first strand 3′ cDNA primer and a first strand 5′ cDNA primer comprising a reference specificity determining box;
• and
• ii) a test sample comprising an amount of test RNA, wherein the test RNA is labeled with the first strand 3′ cDNA primer and a first strand 5′ cDNA primer comprising a test specificity determining box, thereby providing a sample mixture;
• b) subjecting the sample mixture to amplification conditions for polymerase chain reaction, thereby producing an amplified sample mixture;
• c) dividing the amplified sample mixture into a first divided sample mixture and a second divided sample mixture;
• d) subjecting the first divided sample mixture to linear extension conditions, wherein the linear extension conditions comprise using linear extension primers that specifically bind to the reference specificity determining box, thereby producing nucleic acids comprising amplified reference nucleic acid;
• and subjecting the second divided sample mixture to linear extension conditions, wherein the linear extension conditions comprise using linear extension primers that specifically bind to the test specificity determining box, thereby producing nucleic acids comprising amplified cDNA of the test RNA;
• and
• e) determining the amount of test RNA in the test sample, wherein the amount of the test RNA in the test sample correlates with a ratio of the amount of amplified cDNA of the test RNA over the amount of the amplified reference nucleic acid, multiplied by the known amount of the reference nucleic acid in the reference sample.

A method of determining the amount of an RNA transcript of interest in a test sample, comprising:

• a) admixing:
• i) a reference sample, wherein the reference sample comprises a known amount of a reference nucleic acid, wherein the reference nucleic acid is labeled with a first strand 3′ cDNA primer and a first strand 5′ cDNA primer comprising a reference specificity determining box;
• and
• ii) a test sample comprising an amount of test RNA, wherein the test RNA is labeled with the first strand 3′ cDNA primer and a first strand 5′ cDNA primer comprising a test specificity determining box, thereby providing a sample mixture;
• b) subjecting the sample mixture to amplification conditions for polymerase chain reaction, thereby producing an amplified sample mixture;
• c) dividing the amplified sample mixture into a first divided sample mixture and a second divided sample mixture;
• d) subjecting the first divided sample mixture to continued amplification conditions, wherein the continued amplification conditions comprise using continued amplification primers that specifically bind to the reference specificity determining box, thereby producing nucleic acids comprising amplified reference nucleic acid;
• and subjecting the second divided sample mixture to continued amplification conditions, wherein the continued amplification conditions comprise using continued amplification primers that specifically bind to the test specificity determining box, thereby producing nucleic acids comprising amplified cDNA of the test RNA containing amplified RNA transcript of interest;
• and
• e) determining the amount of the RNA transcript of interest in the test sample, wherein the amount of the RNA transcript of interest in the test sample correlates with a ratio of the amount of cDNA of the amplified RNA transcript of interest in the amplified cDNA of the test RNA, over the amount of the amplified reference nucleic acid, multiplied by the known amount of the reference nucleic acid in the reference sample.