Small nucleic acid quantification using split cycle amplification

A method of generating and amplifying cDNA, the method comprising:

• (a) reverse transcribing a target RNA comprising a non-polyA region and a polyA tail by:
• (i) hybridizing a reverse transcription (RT) primer to the target RNA, wherein the RT primer comprises from 5′ to 3′:
• X-(T)m-Y-(T)n-Z
• wherein X is an optional 5′ tail nucleotide sequence of 1-10 nucleotides that is not complementary to the target RNA;
• T is thymine;
• Y is a single nucleotide that is C, G, or A;
• Z is an optional sequence of 1-10 nucleotides that is complementary to a portion of the non-polyA region adjacent to the polyA tail;
• m is 1-20;
• and
• n is 1-20;
• and
• (ii) reverse transcribing the target RNA by extending the RT primer with a RNA-dependent polymerase to generate a cDNA incorporating the RT primer;
• and
• (b) amplifying the cDNA by:
• (i) hybridizing an amplification primer comprising a sequence complementary to the -(T)m-Y-(T)n, and
• (ii) extending the amplification primer with a DNA-dependent polymerase.