The construction of a standard RNA synthesized for quantitative RT-PCR system

S. Fujimaki, T. Funato, J. Fujiwara, J. Satoh, T. Miura, M. Kaku, Y. Tohmiya, T. Sasaki

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)54-59
Number of pages6
JournalRinsho byori. The Japanese journal of clinical pathology
Volume48
Issue number1
Publication statusPublished - 2000 Jan

ASJC Scopus subject areas

  • Medicine(all)

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