Absolute quantitation of nucleic acids by rt-pcr
A method for obtaining a cRNA for use in generating calibration data, e.g., a standard curve, for absolute quantitation of RNA by RT-PCR is disclosed. The method includes the steps of: providing a synthetic oligonucleotide comprising an amplicon, a promoter sequence located 3′ relative to the amplicon; synthesizing complementary RNA (cRNA) by in vitro transcription of the synthetic oligonucleotide; quantitatively assaying the cRNA by an independent method; and generating calibration data using a known quantity of the cRNA.
1 - 18 . (canceled)
19 . A calibrator for absolute quantitation of target RNA by RT-PCR comprising a single cRNA species synthesized by reverse transcription from a synthetic oligonucleotide and quantitatively assayed by an independent method, wherein said synthetic oligonucleotide comprises an amplicon and a promoter sequence located 3′ relative to the amplicon.
20 . The calibrator of claim 19 , wherein said promoter sequence is a bacteriophage promoter sequence.
21 . The calibrator of claim 20 , wherein said promoter sequence is a T7 promoter sequence.
22 . The calibrator of claim 21 , wherein said T7 promoter sequence is CCTATAGTGAGTCGTATTA (SEQ ID NO:1).
23 . The calibrator of claim 19 , wherein said synthetic oligonucleotide further comprises a flanking sequence of 2-20 nucleotides adjacent to the amplicon.
24 . The calibrator of claim 23 , wherein said flanking sequence is 5′ and adjacent to the amplicon.
25 . The calibrator of claim 23 , wherein said flanking sequence is 3′ and adjacent to the amplicon.
26 . The calibrator of claim 23 , wherein synthetic oligonucleotide further comprises both a 5′ and a 3′ flanking sequence adjacent to the amplicon.
27 . The calibrator of claim 23 , wherein said flanking sequence is 8 to 12 nucleotides in length.
28 . The calibrator of claim 19 , wherein said amplicon is 30 to 70 nucleotides in length.
29 . The calibrator of claim 28 , wherein said amplicon is 40 to 60 nucleotides in length.
30 . The calibrator of claim 19 , wherein said synthetic oligonucleotide is 60 to 140 nucleotides in length.
31 . The calibrator of claim 30 , wherein said synthetic oligonucleotide is 70 to 130 nucleotides in length.
32 . The calibrator of claim 31 , wherein said synthetic oligonucleotide is 80 to 120 nucleotides in length.
33 . The calibrator of claim 32 , wherein said synthetic oligonucleotide is 90 to 110 nucleotides in length.
34 . The calibrator of claim 19 , further comprising heterologous RNA.
35 . The calibrator of claim 34 , wherein said heterologous RNA is total RNA.
36 . The calibrator of claim 35 , wherein said heterologous RNA is yeast total RNA.
37 . The calibrator of claim 19 , wherein said cRNA species is MGB.
38 . The calibrator of claim 19 , wherein said cRNA is assayed quantitatively by measuring its absorbance at 260 nm.
39 . A kit comprising the calibrator of claim 19 .
40 . The kit of claim 39 comprising more than one aliquot of said cRNA species.
41 . The kit of claim 40 , wherein each said aliquot comprises successive serial 1:10 dilutions of said cRNA species.