stabilizing solution for tissue
RNAwait™
- preserves RNA in tissue without immediate need to freeze
- one month at 4 °C, one week at 25 °C, one day 37 °C
- maintains RNA integrity (RIN) and molecular RT-qPCR stability
- allows up to 5 freeze-thaw cycles without impact on RNA quality
RNAwait™ is a tissue collection and storage solution that quickly permeates fresh tissues to stabilize RNA. It streamlines operations by eliminating the need for immediate downstream processing or freezing of the sample. Instead, the tissue can be submerged in RNAwait™ and stored. Hence, the RNA extraction step can wait.
RNAwait treatment results in RNA of identical quality to that from fresh tissue
RNA from 6 murine hearts and kidneys was either freshly extracted, or after overnight incubation in RNAwait at 4 °C followed by storage at -80 °C for 3 days and thawed. The integrity (RIN) and Cq value (RT-qPCR, not shown) are similar between the 2 conditions, demonstrating that RNAwait perfectly preserves RNA in tissue.
Perfect RNA integrity (intact 18S and 28S ribosomal RNA) from fresh tissue (lane 1,2) and RNAwait treated tissue (3,4) for murine heart (1,3) and kidney (2,4).
Freeze-thaw cycles do not affect RNA quality
Murine heart and kidney tissues (n=6) were incubated overnight at 4 °C in RNAwait, followed by RNAwait removal and 5 repeated freeze (-20 °C, 1 day) and thaw (25 °C, 1 hour) cycles. The resulting RNA quality in terms of RT-qPCR Cq value and RIN value is similar to that from fresh tissues.
Left: Identical RT-qPCR Cq value distribution of Cd81 transcripts in murine heart and kidney (n=6) from fresh tissue (blue) or stabilized in RNAwait (pink) followed by 5 freeze-thaw cycles.
Right: Perfect RNA integrity in RNAwait treated kidney and heart tissue after 5 freeze-thaw cycles.
High RNA quality in tissue stored at ambient temperature
Murine heart and kidney tissues (n=48) were incubated overnight at 4 °C in 3 different lots of RNAwait, followed by continued storage at either 4 °C for 30 days (n=12), 25 °C for 7 days (n=12), or 37 °C for 1 day.
After these prolonged incubations, RNAwait was removed, and tissues stored at -20 °C for at least 2 weeks, followed by thawing, RNA extraction, and RT-qPCR (12 ng RNA input).
As a comparator, RNA was extracted from fresh hearts and kidneys (n=12). The RNA integrity (RIN, not shown) and molecular stability (RT-qPCR Cq value) is perfectly preserved across these non-freezing storage conditions.
Bar chart of RT-qPCR Cq values of Anapc5 and Cd81 in murine heart and kidney tissue (n=12) stored at 3 different storage conditions, each using 3 different lots of RNAwait. Error bars denote standard errors of the mean.
Preservation of histology
Histological examination of FFPE-embedded tissue after RNAwait preservation allows recognition of the characteristic tissue structures.
Hematoxylin and eosin (H&E) staining of a formalin-fixed and paraffin-embedded fresh kidney tissue (left) or an RNAwait-treated (overnight at 4 °C) kidney tissue (right).
Dried blood spot RNA preservation
While RNAwait was purposely developed to stabilize RNA in tissue, it also stabilizes RNA in capillary blood collected on pre-treated cards. Upon proper storage of the card with blood (using desiccant), RNA remains intact (high RIN values) at 4 °C for at least 2 weeks, at 25 °C for 3 days, or at -20 °C / -80 °C for much more extended periods.
RNAwait pretreated cards result in stable RT-qPCR results (FOS and IL1B) for at least 2 weeks when stored at 4 °C, for 8 days at 25 °C, and for extended periods at -20 °C / -80 °C. See the Download section for an RNA extraction SOP and additional details.
