Figure 5. Fixation and storage introduces major gene-to-gene variations in qRT-PCR.

Aliquots of a human liver sample were cryopreserved or fixed in formalin and paraffin embedded. (A) RNA was extracted from samples at different timepoints including technical replicates. (B) Comparison of qRT-PCR data for 92 genes from cryopreserved and FFPE human liver samples reveals an average difference of the ct values ranging from 4 cycles (6 months) to 8 cycles (1 year) increasing with storage time at room temperature. Extraction from the FFPE sample which had been stored for one year was done in duplicates. cDNA generation was performed in duplicates from the same RNA, and qRT-PCR was performed twice from the same cDNA. Data was generated with the TaqMan “Human Molecular Mechanisms of Cancer” assay, individual ct values are shown.