Fig 1.
Schematic overview of the study setup.
Human clinical or rat tissues were freshly collected, aliquoted and either directly snap-frozen (Cryo), or fixed in 4% neutral buffered formaldehyde (NBF) or in PAXgene Tissue Fix and Stabilizer. Paraffin embedded tissue blocks (PET) were stored under controlled temperatures for up to 7 years in case of human and up to 9 years in case of rat tissues until RNA and DNA extraction.
Fig 2.
RNA integrity from human PET blocks decreases depending on storage temperature.
Analysis of RNA integrity number (RIN) was performed from human PFPE and FFPE tissues using an Agilent Bioanalyzer. PET blocks of different tissue types were stored at room temperature (RT) and 4°C prior to RNA extraction. RNA was extracted from 8 different cases including four different tissue types (soft tissue, stomach, duodenum, liver) at several time points for up to 84 months (m) of storage (S1 Table). For each time point, fixation method and storage condition the mean RIN value with standard deviation was calculated (total RIN values n = 411).
Fig 3.
RNA integrity from rat PET blocks decrease depending on storage temperature.
For each of five different tissue types (liver, kidney, spleen, lung and intestine) and four different temperatures (22°C, 4°C, -20°C, and -80°C) 3 blocks of FFPE and PFPE rat tissue were stored. RNA was extracted from block 1 after 3, 6 and 12 months (m), from block 2 after 24, 36, 48 and 72 m and from block 3 after 108 m of storage. Mean RNA integrity number (RIN) values with standard deviation from FFPE (A) and PFPE tissue (B) are shown for triplicate extractions from five different tissue types for each fixation method, storage time point and temperature (total RIN values n = 942). Dotted line indicates block change.
Fig 4.
RT-qPCR-performance is independent from storage time in PET blocks stored at 4°C.
Human FFPE (A and C) and PFPE (B and D) tissue (case #1, soft tissue) were stored at 4°C (A and B) or at room temperature (C and D) for up to 6 years prior to RNA extraction. Amplification of human GAPDH gene fragments ranging from 71 to 323 base pairs (bp) was performed in SYBR-Green RT-qPCR assays. Missing data points represent no specific amplification possible.
Fig 5.
Improved RT-qPCR-performance of RNA from human tissue blocks stored at 4°C.
RT-qPCR-performance of RNA from human FFPE and PFPE tissues stored prior to RNA extraction at 4°C or at room temperature (RT) for up to 7 years. Mean delta Ct values with standard deviation of two assays with different amplicon lengths of human GAPDH gene (Ct 200 bp–Ct 71 bp) from up to 8 cases are shown. Statistically significant differences (P < 0.05) between the first (1-3m) and following timepoints of RT storage are shown. No significant differences were found in case of storage at 4°C.
Fig 6.
Improved RT-qPCR-performance of RNA from rat tissue blocks stored at 4°C or frozen for 9 years.
PFPE and FFPE tissues were stored 108 months (m) at different temperatures (22°C, 4°C, -20°C, and -80°C). Reverse transcription and amplification in five different rat ACTB SYBR-Green RT-qPCR assays ranging from 109 to 438 base pairs (bp). Mean Ct values with standard deviation from triplicate extractions from each of five different tissues (liver, kidney, spleen, lung and intestine) are shown for each assay. RNA from cryo preserved tissue was used as a reference, shown as dashed line.
Fig 7.
Storage of PET blocks for 9 years at lower temperatures prevents from DNA degradation.
Analysis of DNA integrity from FFPE (A) and PFPE (B) tissues of rat liver, kidney, spleen, lung and intestine on Agilent 4200 TapeStation system with genomic DNA Analysis ScreenTape assay. PET blocks were stored prior to DNA extraction for 108 months at 22°C, 4°C, and -20°C. DNA was extracted from 3x 10μm sections in triplicates.
Table 1.
Average DNA molecule size from rat PET blocks stored at different temperatures for 9 years.
Fig 8.
Improved qPCR-performance of DNA from PET blocks stored for 9 years at lower temperatures.
FFPE and PFPE animal tissues were stored prior to DNA extraction for 108 months at different temperatures (22°C, 4°C, and -20°C). qPCR was performed with four different SYBR-Green qPCR assays of the rat ACTB gene with amplicon length of 271, 523, 650 and 747 base pairs (bp). Triplicate extractions from each PET block were amplified. DNA from cryo-preserved rat tissue was used as reference, shown as dashed line. Mean Ct values with standard deviation from triplicate extractions from each of five different tissues (liver, kidney, spleen, lung and intestine) are shown for each assay.