Chemical Metrology and Biometry DepartmentChemical Metrology and Biometry Department

National Institute of Metrology (Thailand)National Institute of Metrology (Thailand)

CCQM P103.1CCQM P103.1

““Measurement of multiple RNA transcripts”Measurement of multiple RNA transcripts”

Duangkamol ViroonudompholDuangkamol Viroonudomphol

Chaiwat PrawettongsoponChaiwat Prawettongsopon

2

Real-time PCR 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

ERCC measurementERCC measurement: : Absolute quantificationAbsolute quantification

4

Transcript U1 copies number U2 copies number Relative expression Ratio

(U1/U2)

Expanded uncertainty

ERCC-13 47173.04 68255.49 0.69 0.22

ERCC-25 5287.89 5258.24 1.01 0.28

ERCC-42 986.5 7285 0.14 0.04

ERCC-99 50061.44 57207.59 0.88 0.33

ERCC-113 20347.24 7070.39 2.88 0.95

ERCC-171 88692.57 17350.62 5.11 1.73

Optional GOIs: Optional GOIs: Relative quantificationRelative quantification

-Based on a doubling of template with each PCR cycle 2 -∆Cq(U1-U2)

-Normalised with CASC3 GOIs/CASC3

Transcript Relative expression Ratio (U1/U2) Expanded uncertainty

MMP1 42.65 3.76

NES 1.47 0.12

SLC1A3 12.95 1.72

6

Source of uncertainty budgetsSource of uncertainty budgets

ERCCs: Regression from the calibrantion, Precision, Pipetting bias

GOIs: Precision, Pipetting bias

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

ua = a √ (ub/b)2 + (uc/c)2

7