[A] Genomic DNA was purified from various FFPE rat tissues that were stored at room temperature for the times indicated. Purification was performed using either the AllPrep DNA/RNA FFPE Kit or, as a control, the QIAamp DNA FFPE Tissue Kit (a dedicated kit for DNA purification from FFPE samples) both including RNase digestion. DNA yields from 20 μm sections of each sample were determined by absorbance measurement. [B] RNA was purified from various FFPE rat tissues that were stored at room temperature for the times indicated. Purification was performed using either the AllPrep DNA/RNA FFPE Kit or, as a control, the RNeasy FFPE Kit (a dedicated kit for RNA purification from FFPE samples). RNA yields from 10 μm sections of each sample were determined by absorbance measurement. The AllPrep Kit performed just as well as the dedicated DNA/RNA purification kits in recovering DNA/RNA from FFPE samples.
Total RNA was purified from human breast FFPE tissue using the AllPrep DNA/RNA FFPE Kit. RNA was then reverse-transcribed using RT2 FFPE PreAMP technology. Gene expression analysis by real-time PCR was performed using the Human Cell Cycle RT2 Profiler PCR Array, comparing a tumor sample to a nontumor sample. ΔΔCT analysis shows the x-fold difference in gene expression of tumor sample compared to nontumor sample.
[A] DNA and [B], [C] RNA were purified from various FFPE rat tissues using either the AllPrep DNA/RNA FFPE Kit or, as a control, dedicated kits for DNA or RNA purification from FFPE samples (QIAamp DNA FFPE Tissue Kit or RNeasy FFPE Kit). Real-time PCR or real-time RT-PCR was carried out on an ABI PRISM 7900HT Sequence Detection System using [A] the QuantiTect SYBR® Green PCR Kit to analyze a 78 bp amplicon of the Prnp gene or [B], [C] the QuantiTect SYBR® Green RT-PCR Kit to analyze Jun oncogene expression. The AllPrep Kit and the dedicated kits provided comparable CT values, indicating that all kits achieved similar efficiency in recovering usable DNA or RNA. [C] In addition, analysis of Jun expression was carried out without reverse transcriptase (-RT). The amplification plot for the spleen sample, a DNA-rich tissue, indicated the virtual absence of genomic DNA contamination.
