1. Chemical Metrology and Biometry Department National Institute of Metrology (Thailand) CCQM P103.1 “Measurement of multiple RNA transcripts” Duangkamol Viroonudomphol Chaiwat Prawettongsopon

2. 2 Real-time PCR Instrument: ABI 7500 Real Time PCR System RT-qPCR kit: AgPath-ID RT-PCR Reagents kit (ABI) Reaction:10 µl, RNA sample = 1 µl/reaction Analysis software: 7500 Software V 2.0.6

3. ERCC measurement : Absolute quantification

4. 4 TranscriptU1 copies numberU2 copies numberRelative expression Ratio (U1/U2) Expanded uncertainty ERCC-13 47173.0468255.490.690.22 ERCC-25 5287.895258.241.010.28 ERCC-42 986.572850.140.04 ERCC-99 50061.4457207.590.880.33 ERCC-113 20347.247070.392.880.95 ERCC-171 88692.5717350.625.111.73

5. Optional GOIs: Relative quantification -Based on a doubling of template with each PCR cycle 2 -∆Cq(U1-U2) -Normalised with CASC3 GOIs/CASC3 TranscriptRelative expression Ratio (U1/U2)Expanded uncertainty MMP1 42.653.76 NES 1.470.12 SLC1A3 12.951.72

6. 6 Source of uncertainty budgets ERCCs: Regression from the calibrantion, Precision, Pipetting bias GOIs: Precision, Pipetting bias Relative expression ratio (a) = U1/U2 = b/c u a = a √ (u b /b) 2 + (u c /c) 2

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