Poor Ct reproducibility — high within-group variability (qPCR (RT-qPCR))

Question

Accepted Answer

Technical replicates of the same sample have SD > 0.5 cycles; standard curve linearity is poor; loading order influences results. Common causes: 1) Pipetting error (different volumes between wells) 2) Pipette out of calibration 3) Master mix not adequately mixed 4) Sample concentration uneven across wells 5) Evaporation or air bubbles distort signal Solutions: 1) Use a calibrated pipette and ideally a multichannel for replicates 2) Pipette carefully, avoid air bubbles, change tips between samples 3) Vortex master mix gently to mix; spin down before pipetting 4) Add samples in random rather than fixed order to detect position effects 5) Increase replicate count (≥ 3 technical) and seal plate properly to prevent evaporation