[The construction of a standard RNA synthesized for quantitative RT-PCR system].
We constructed the standard RNA synthesized for the chimeric AML1-MTG8 transcripts and the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase(GAPDH) transcripts in real-time quantitative RT-PCR system. AML-MTG8 transcripts was detectable in 10 fg of synthetic RNA(3.5 x 10(3) copies). Linearity was from 3.5 x 10(3) to 3.5 x 10(9) copies. Threshold cycle(CT) is defined as the fractional cycle number at which the reporter fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. The standard curve, where the known amounts of RNAs were used, showed a good correlation between the copies of AML1-MTG8 RNA and CT(r = -0.995). The within-run and day-to-day coefficients of variation(CV) in AML1-MTG8 RNA by this system were 9.5-24.7%(n = 10) and 21.7-42.2% (n = 8), respectively. GAPDH transcripts was detectable in 10 fg of synthetic RNA(6.1 x 10(4) copies). Linearity was from 6.1 x 10(4) to 6.1 x 10(8) copies. The standard curve, where the known amounts of RNAs were used, showed a good correlation between the copies of GAPDH RNA and CT(r = -0.993). The within-run and day-to-day CV in GAPDH RNA by this system were 9.3-14.6%(n = 10) and 14.7-15.8% (n = 10), respectively. Thus, we suggested that synthesized RNA as a standard RNA may be useful in quantitative RT-PCR for clinical application.
Fujimaki
S
S
Clinical Laboratory, Tohoku University Hospital, Sendai.
Funato
T
T
Fujiwara
J
J
Satoh
J
J
Miura
T
T
Kaku
M
M
Tohmiya
Y
Y
Sasaki
T
T
D050676:Core Binding Factor Alpha 2 Subunit; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000592:standards*; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors / Q000235:genetics
C107844:AML1-ETO fusion protein, human; D050676:Core Binding Factor Alpha 2 Subunit; D015514:Oncogene Proteins, Fusion; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases