Recently, we gave a series of webinars covering the different challenges and possible solutions for getting reliable gene expression profiles, with a focus on RNA extraction and stabilization from challenging samples. During these webinars, the audience had a variety of interesting questions. We would like to share with you our answers to the top 9 questions to help you with your own research.
1. How can I avoid little or no RNA yield when using an RNeasy Kit?
RNA in tissues is not protected after harvesting and is prone to degradation. To minimize this, ensure your samples are handled and stored correctly before using the RNeasy Kits. Using a stabilizing reagent such as RNAlater, flash freezing in liquid nitrogen, or disrupting and homogenizing in the presence of RNase-inhibiting or -denaturing reagents are all suitable methods for protecting RNA. Store samples at –70°C, and do not allow tissue samples to thaw during handling or weighing. Cell pellets can be partially thawed and flicked to dislodge the pellet. Downstream procedures should be carried out as quickly as possible. Samples can also be stored at –70°C in lysis buffer (Buffer RLT) after disruption and homogenization. Frozen samples are stable for several months.
For optimal RNA yields using RNeasy Kits, it is crucial to:
(a) efficiently disrupt and homogenize the starting material
(b) use the correct amount of starting material – do not overload!
(c) perform all protocol steps at room temperature
(d) perform a dry-spin prior to elution, as described in the relevant protocol, for a full 5 minutes
(e) prepare the 80% ethanol for the wash steps with RNase-free water only
(f) dispense the RNase-free water for elution onto the center of the membrane
(g) optionally repeat the elution step, and incubate the spin column on the bench for 10 minutes with RNase-free water before centrifuging
2. Can cells stabilized with RNAprotect Cell Reagent be used for cell sorting by flow cytometry (FACS)?
We do not recommend using 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.
3. Can I stabilize previously stored isolated RNA by adding any stabilization reagent, such as RNAlater?
We do not recommend storing previously isolated RNA in RNAlater Stabilization Reagent, since it will be very difficult to recover isolated RNA from it. Purified RNA can be stored at –20°C or –70°C in sterile RNase-free water. Under these conditions, we found no detectable RNA degradation after 1 year of storage.
4. Is it possible to perform laser captured microdissection with RNAlater-stabilized tissue?
For laser captured microdissection, it’s best to either flash-freeze the tissue and do cryosectioning or to use FFPE material.
5. Do you have a kit for RNA isolation from any kind of sample type?
The RNeasy Plus Universal Kit enables single-tube purification of RNA from any animal or human tissue sample, including difficult-to-lyse fibrous and fatty tissues. For high-throughput purification, the RNeasy 96 Universal Tissue Kit is also available.
Please refer to the Selection guide for RNA isolation for all sample types to find the optimal solution for you.
6. What is the recommended incubation time to stabilize tissue RNA using RNAlater?
The RNAlater RNA Stabilization 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 incubating 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 RNAlater 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 outlined in step 5 of the “Protocol for RNA Stabilization with RNAlater RNA Stabilization Reagent” in the RNAlater Handbook.
7. Can RNAlater be used to stabilize tissue when it has already been flash-frozen?
No, we do not recommend using RNAlater to stabilize flash-frozen tissue. It should only be used to stabilize fresh tissue.
8. Can I stabilize adipose tissue using RNAlater prior to RNA isolation?
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 RNAlater reagent to permeate into the tissue and sufficiently stabilize it.
9. I accidentally added RNAlater to my cells and now the cells are difficult to pellet. What can I do?
If the cells in RNAlater cannot be collected by centrifugation, please try one of the following suggestions:
a) 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, as outlined in step 5 of the protocol.
b) If cells are floating on the surface of the RNAlater reagent, try removing the reagent by pipetting from underneath. Leave behind approximately 100 μl of RNAlater, and add 350 μl Buffer RLT before proceeding with the protocol “RNeasy Mini Protocol for Isolation of Total RNA from Animal Cells.” For every 100 μl of cells in RNAlater, use 250 μl of 96–100% ethanol instead of the 70% ethanol listed in step 4 of the standard protocol.
c) Dilute the sample 10x by adding cold PBS. Pellet cells by centrifugation. Caution: Cells may lyse.
d) 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 RNAprotect Cell Reagent.