RNAprotect Reagents and Tubes

RNAprotect Tissue Reagent is recommended for stabilization and protection of RNA in animal tissues. It is not recommended for cultured cells because it may be difficult to pellet and remove them from the reagent prior to RNA isolation (see FAQ 364). 

Instead, we recommend to use the RNAprotect Cell Reagent.  This reagent is for immediate RNA stabilization in sorted or cultured cells with no need to remove medium

For Laser Captured Microdissection, its best to either flash-freeze the tissue and do cryosectioning, or to use FFPE material. 

In general, this is possible and has been done successfully in our labs with brain tissue. However, if the starting material for RNA isolation is extremely fatty, it will be difficult for the aqueous RNAprotect Tissue Reagent to permeate into the tissue and therefore, to sufficiently stabilize it.

Yes, RNAprotect Cell Reagent of the RNeasy Protect Cell Mini Kit can be purchased separately in a bulk size of 250 ml for processing large cell culture volumes.

The RNAprotect Tissue Reagent penetrates the sample by diffusion and protects RNA immediately upon contact with the surface layer and outer portions of solid tissues. In order to ensure reliable stabilization of RNA even in the inner parts of solid tissues, we recommend incubation for at least 45 minutes in the reagent prior to RNA isolation using RNeasy Kits. Note that tissue size is critical for successful RNA stabilization with RNAprotect technology. Samples must be cut into slices less than 0.5 cm thick for rapid and reliable stabilization in interior tissue parts. RNA degradation will occur in tissue slices that are too thick.

For archival storage in the freezer, first incubate the sample overnight in the reagent at 2–8°C. Please see detailed instructions for storage under step 5 of the “Protocol: Stabilization of RNA in Harvested Animal Tissues” in the RNAprotect Handbook.

No, we do not recommend to use the RNAprotect Tissue Reagent to stabilize flash-frozen tissue. It should only be used to stabilize fresh tissue.

Yes, please follow the User-Developed Protocol 'Preparation of RNAprotect preserved tissues for pathological studies' (PROM-15329).

We do not recommend to store previously isolated RNA in RNAprotect Tissue Reagent, since it will be very difficult to recover isolated RNA from it. Purified RNA can be stored at −30 to −15°C or −90 to −65°C in sterile RNase-free water. Under these conditions, we found no detectable RNA degradation after 1 year of storage.

Different samples of varying water content may affect the point at which the reagent will form a solid at low temperature. Other factors can be temperature variation from freezer to freezer, or even within the same freezer (e.g., different temperatures near the door and in the back region of the freezer). Regardless, the performance of the RNAprotect Tissue Reagent and the RNA quality are not affected by freezing of the reagent.

Yes, but you have to make sure that the RNAprotect Bacteria Reagent is diluted in PBS or water prior to use (2 volumes reagent : 1 volume PBS or water) in order to achieve its optimal working concentration. The diluted reagent is directly added to the bacteria growing on the solid substrate, and reagent and bacteria are mixed thoroughly. The reagent is not recommended for stab cultures, since sufficient access of the reagent to all bacteria for immediate RNA stabilization cannot be guaranteed.

For RNA isolated on the BioRobot EZ1 and BioRobot M48:

The RNA can be directly applied to the Agilent Bioanalyzer, since it is being denatured in the final protocol steps of these isolation procedures.

For RNA prepared with all other QIAGEN RNA Isolation Products:

We recommend to denature the samples in a water bath for 2 min at 70°C, and then place them directly on ice prior to loading them onto the Agilent Bioanalyzer.

If the cells in RNAprotect Tissue Reagent cannot be collected by centrifugation, please try one of the following suggestions:

1) Add 600 µl Buffer RLT to a maximum of 200 µl sample volume, and proceed with step 3 of the "RNeasy Mini Protocol for Isolation of Total RNA from Animal Cells" in the RNeasy Mini Handbook. Load the lysate onto the column in successive aliquots in step 5 of the protocol.

2) If cells are floating on the surface of the RNAprotect Tissue Reagent, try removing the reagent by pipetting from underneath. Leave behind approximately 100 ul of RNAprotect Tissue Reagent, and add 350 ul Buffer RLT before proceeding with the protocol "RNeasy Mini Protocol for Isolation of Total RNA from Animal Cells". For every 100 ul of cells in RNAprotect Tissue Reagent, use 250 ul of 96-100% ethanol instead of the 70% ethanol listed in step 4 of the standard protocol.

3) Dilute the sample 10x by adding cold PBS. Pellet cells by centrifugation. Caution: Cells might lyse.

4) Try to pipette the floating cells off the surface.

Note that the above steps are suggestions, rather than official protocol recommendations. Please try a "pilot" run on a test sample first.

For RNA stabilization of cells, we recommend using the RNAprotect Cell Reagent.

Tissues preserved in RNAprotect Tissue Reagent can be stored as long as listed below:

We never have seen it in-house for mouse or rat pancreas, but if a lot of fatty tissue is present in the tissue sample it may float on top. Our R&D department has obtained good results with RNAprotect Tissue Reagent preserved mouse and rat pancreas.

We do not recommend to use cells stabilized with RNAprotect Cell Reagent for cell sorting as cell morphology will be altered after adding the reagent, and stabilized cells may lyse during the sorting process.