Download FACS RNA

FAC-Sorting for RNA Isolation
Per Dr Blazar please meet with Mark Osborn ([email protected]) to discuss this work plan prior to scheduling FACS time for RNA isolation. References:
Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence
from GFP but not from DsRed
BMC Research Notes 2010, 3:328 Fluorescence activated cell sorting (FACS) using RNAlater to minimize RNA degradation
and perturbation of mRNA expression from cells involved in initial host microbe
interactions
Journal of Microbiological Methods Volume 70, Issue 1 July 2007, Pages 205–208
DAY-­‐1 Preparation. CRITICAL NOTE: IT IS ESSENTIAL TO WORK WITH RNase FREE MATERIALS DURING TISSUE HARVEST AND PROCESSING. Any materials that have been autoclaved are contaminated with RNases that can affect downstream applications. The use of RNase-­‐free BSA in place of serum in sample buffers is preferred as some serum sources may be inherently contaminated with RNases (see following note regarding acetylated and non-­‐acetylated BSA). Affymetrix states: Many nucleases are irreversibly inactivated by acetylation, but the stabilizing action of BSA is unaffected by this procedure.(1,2) Thus, acetylation ensures that the use of albumin does not introduce additional nucleases to reaction mixture. (acetylated BSA (Affymetrix catalog 10848): Additionally, Life Technologies states: BSA preparations are acetylated to inactivate contaminating nucleases, particularly RNase. However, acetylation also modifies the BSA. While acetylated BSA functions in restriction digestions, the acetylation process changes the binding characteristics of BSA as a blocking agent. Non-­‐
acetylated BSA can be used when DNA or RNA integrity is critical.: Ultrapure BSA Non-­‐Acetylated (Life Technologies: Catalog Number AM2616, AM2618). THEREFORE, A CAREFUL COST ANALYSIS SHOULD BE DONE TO DETERMINE WHICH BSA IS ADVOCATED. Only filtered, certified nuclease free pipettes should be used. FACS buffer should be made in tissue culture grade plastic ware (eg Millipore Stericup, UStores catalog: CX11776) Work station, instruments, mortar, and pestle (if used) should be wiped with RNase Away, wrapped in sealable plastic autoclave receptacles available from dishwashing, and then autoclaved. In between animals and tissues the instruments should be wiped with RNase Away wipes. When aspirating supernatant it is advisable to either pour off the media or remove the cotton plug from a sterile, nuclease free, tissue culture pipette and attach it to the vacuum line and use it. Alternatively, a glass pipette can be attached to the vacuum line and then placed into a P1000 tip box containing unfiltered, nuclease free tips to allow the suction to attach the P1000 to the glass pipette. This can be used to aspirate media in a nuclease free manner. DAY 0. Harvest. CRITICAL NOTE: It is essential to harvest tissue and place them IMMEDIATLEY in either RNAlater for archival or RNA isolation buffer (e.g Trizol). For most tissues either a 0.5x0.5x0.5 or 1x1x1 cm block of tissue is sufficient for high yield RNA for downstream applications and can be added to 1 mL of Trizol or RNA later. Samples in RNAlater can be archived long term. Trizol can be prepared in 2 mL screw top vials or Miltenyi M-­‐tubes and homogenized by a hand-­‐held homogenizer or beads for the former or the Gentle MACS Dissociater (See Note 1). Homogenized Trizol can then be utilized to isolate total RNA using the RNA isolation procedure in Note 2. Each animal should be processed and the tissues placed in the RNA buffer in <5 minutes to prevent RNA degradation. Once in RNA later or homogenized in Trizol the tissues are stable long term at -­‐80 C. Tissues homogenized in Trizol can be processed immediately as well (Note 2) and purified RNA should be stored at -­‐80 C. Cell Sorting: Critical Note: Intracellular staining/cellular permeabilization: Fixation of cells
using formalin and other aldehydes should be avoided because it causes nucleic acid
cross-linking and contributes to RNA degradation. Although ethanol fixation does not
negatively affect RNA, it does cause cell membrane permeability and possible mRNA
leakage. For these reasons, it is preferable to use cell surface markers in order to avoid
cell fixation–permeabilization before FACS for optimal RNA yield and integrity.
-­‐when possible, antibodies (for surface staining) that are utilized only under nuclease free conditions should be used for cell staining. Cells/tissue should remain on ice during staining. All washes should be performed as detailed above with nuclease free reagents. The best quality RNA is obtained with minimal sort times and rapid preservation of the cells in Trizol (cells sorted into RNase free media can be spun, the supernatant removed, and Trizol added to the cell pellet followed by archival at -­‐80 C). For extended sorting of rare populations cells can be sorted directly into RNAlater as follows: The optimal volume of RNAlater and limit of dilution is 10%. Therefore, 15 mL conical tubes containing 9 mL of RNAlater should be provided to the Flow Core and 1 mL of sorted material may be added to each. If one sample is predicted to need more than 1 mL then multiple tubes should be prepared and pooled as described subsequently. Note: Suggested procedures for single, multiple, and low cell yield situations are provided. For Single Collection Tube: Centrifuge 15 mL conical tube at 2500 ×g for 30 min. RNAlater can then be CAREFULLY and completely removed leaving only the cell pellet in the tube. Trizol reagent is then added directly to the cell pellet and RNA is isolated following the instructions in Note 2. For Multiple Collection Tubes that then must be pooled (eg one treatment group that requires multiple collection tubes): Centrifuge 15 mL conical tubes at 2500 ×g for 30 min. After centrifugation, RNAlater is partially removed leaving approximately 500 μl of volume in the tube, appropriate tubes sets are pooled together, and centrifuged again for 30 min at 2500 ×g. RNAlater can then be CAREFULLY and completely removed leaving only the cell pellet in the tube. Trizol reagent is then added directly to the cell pellet and RNA is isolated following the instructions in Note 2. For Low Cell Yields. Collect sample in RNAlater as detailed above. Centrifuge 15 mL conical tube at 2500 ×g for 30 min. RNAlater is partially removed leaving approximately 500 μl of volume in the tube(s), appropriate tubes sets are pooled together into a screw top vial and spun at maximum speed for 1 minute in a table top Eppendorf centrifuge. RNAlater can then be CAREFULLY and completely removed leaving only the cell pellet in the tube. Trizol reagent is then added directly to the cell pellet and RNA is isolated following the instructions in Note 2. NOTE: This procedure has been used successfully; however, RNA quality cannot be guaranteed due to the high-­‐speed final spin and should be used with that understanding. *****Further questions can be directed to Mark or Chris. Either are able to discuss this in detail prior to any experiments***** Note 1: Also available at: http://www.miltenyibiotec.com/en/PG_1128_764_gentleMACS_M_Tubes.aspx Note 2. RNA Isolation