chemical metrology and biometry department national institute of metrology (thailand)

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CCQM P103.1 Measurement of multiple RNA transcripts. Duangkamol Viroonudomphol Chaiwat Prawettongsopon. Chemical Metrology and Biometry Department National Institute of Metrology (Thailand). Real-time PCR . Instrument: ABI 7500 Real Time PCR System - PowerPoint PPT Presentation

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  • Chemical Metrology and Biometry DepartmentNational Institute of Metrology (Thailand)CCQM P103.1Measurement of multiple RNA transcriptsDuangkamol ViroonudompholChaiwat Prawettongsopon

  • *Real-time PCR Instrument: ABI 7500 Real Time PCR SystemRT-qPCR kit: AgPath-ID RT-PCR Reagents kit (ABI)Reaction:10 l, RNA sample = 1 l/reactionAnalysis software: 7500 Software V 2.0.6

  • ERCC measurement: Absolute quantification

  • *

    TranscriptU1 copies numberU2 copies numberRelative expression Ratio (U1/U2)Expanded uncertaintyERCC-1347173.0468255.490.690.22ERCC-255287.895258.241.010.28ERCC-42986.572850.140.04ERCC-9950061.4457207.590.880.33ERCC-11320347.247070.392.880.95ERCC-17188692.5717350.625.111.73

  • Optional GOIs: Relative quantificationBased on a doubling of template with each PCR cycle 2 -Cq(U1-U2)Normalised with CASC3 GOIs/CASC3

    TranscriptRelative expression Ratio (U1/U2)Expanded uncertaintyMMP142.653.76NES1.470.12SLC1A312.951.72

  • *Source of uncertainty budgetsERCCs: Regression from the calibrantion, Precision, Pipetting biasGOIs: Precision, Pipetting bias

    Relative expression ratio (a) = U1/U2 = b/c

    ua = a (ub/b)2 + (uc/c)2

  • *

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