1. Start with healthy cells.
Starting cells must be healthy. For cell lines, we recommend cells be in the exponential growth phase. DO NOT use confluent or overly concentrated cells. Unhealthy cells are more susceptible to cell lysis in processing, resulting in cell loss.
2. Keep the samples on ice during cell preparation and antibody staining.
It is critical to keep the samples cold during processing to preserve the RNA integrity. We highly recommend adding RNase inhibitors in the buffers used for tissue dissociation and antibody staining to prevent RNA degradation which will lead to a drop in signal. Assay kits contain sufficient RNase inhibitor to process samples according to the protocol. If additional inhibitor is needed for dissociation or staining buffers, it should be purchased separately (Affymetrix cat. No. 71571).
3. Use a swinging bucket centrifuge.
We do not recommend using a fixed-angle centrifuge, as it may result in severe cell loss.
4. Validate the temperature of incubator and use a metal block during hybridization.
Incubator must be validated to hold a temperature of 40˚C +/- 1 ˚C with the QuantiGene Temperature Validation Kit (Affymetrix cat. no. QV0523) before beginning the assay.
A metal heat block for 1.5-mL microfuge tubes should be used in all hybridization steps to ensure uniform hybridization temperature.
5. Perform fixation and permeabilization in the provided 1.5-mL microfuge tubes or polypropylene conical tubes.
Fixation and permeabilization can be performed in any tube. After bulk fixation and permeabilization, all subsequent hybridization steps MUST be performed in the provided 1.5mL microfuge tubes. DO NOT use polystyrene tubes such as FACS tube for fixation/permeabilization/hybridization (e.g. BD Falcon cat. no. 352052) which will cause poor signal.
6. Pipet assay reagents directly into the sample.
When adding Target Probe, PreAmp, Amp and Label Probe solutions to the samples, pipet the reagents directly into the 100 µL- sample and then vortex briefly to mix well. DO NOT pipet reagents against the wall of the tube to allow the reagents drip down into the cell samples.
7. Fluorochrome compatibility:
Organic fluorochromes are compatible with this assay kit, such as FITC, eFluor® 450, eFluor® 660, and Alexa Fluor® 700. BV dyes are also reported to work.
Most protein-based fluorochromes are compatible with this PrimeFlow® RNA Assay Kit, including PE, PE-eFluor® 610, PE-Cyanine5, PE-Cyanine5.5, PE-Cyanine7, APC, and APC-eFluor® 780.
PerCP, PerCP-Cyanine5.5, and PerCP-eFluor® 710 may not be used. We recommend using PE-Cyanine5 or PE-Cyanine5.5 instead.
Qdot® nanocrystal and eVolveTM -antibody conjugates are not compatible with this assay.
Protect samples from light after they have been stained with fluorochrome-conjugated antibodies and labeled for RNA.
8. Intracellular antibody staining compatibility:
The fixation and permeabilization buffers in this PrimeFlow® RNA Assay Kit are compatible with most of eBioscience’s antibodies used for intracellular staining. Some changes in performance and resolution are expected when compared to performance in the Intracellular Fixation & Permeabilization Buffer Set (Cat. No. 88-8824) or Foxp3/Transcription Factor Staining Buffer Set (Cat. No. 00-5523).
The fixation and permeabilization buffers in this kit are compatible with most of eBioscience’s phospho-specific antibodies; however, phospho-specific antibodies that will work only in the IC Fixation/Methanol protocol are not compatible. Please refer to the Technical Support webpage and the Phospho Flow Cytometry Antibody Clone Buffer Selection Guide or the datasheet for the individual antibody.
9. Include appropriate controls.
As with any multi-color panel, controls for instrument set up and for experiment are essential.
Compensation: You must have single-color compensation controls for both antibody staining and RNA staining. If you are using a universal negative for compensation, this sample and all other single-color samples must have gone through all steps of the FlowRNA assay.
FMO: Fluorescence-Minus One (FMO) controls, which is a sample that contains all but one of the fluorochromes being used, is critical for determining whether compensation was set correctly and will assist in understanding background and auto-fluorescence on gated events.
Negative controls; Include an unstained sample that has gone through the entire FlowRNA protocol, minus target probe. No target probe controls are necessary for assessing background associated with the amplification steps. Isotype controls are necessary for assessing the background of antibody staining on cells.
Positive controls: Positive control target probes are necessary to know that they hybridization/amplification steps worked and can be included as one of the three RNA transcripts or as a separate sample.
Biological controls: Whenever possible, biological controls (e.g. unstimulated or uninfected samples) are necessary for assessing the specificity of the target probes.
10. Set up proper compensation controls with the same fluorochromes
Type 1, Type 4, and Type 6 Positive Control Probe Sets must be used to set compensation for RNA signal. DO NOT substitute other fluorochromes which have similar excitation/emission properties. For example, APC should not be used to compensate for Type 1 probes (Alexa Fluor® 647). If antibody staining will be included, single stain controls for each fluorochrome should be included.
Use calibration beads (e.g. Spherotech cat. no. URCP URCP-38-2K) to check the laser alignment.
Two lasers, blue (488 nm) and red (633 nm, or similar), as well as filter sets for FITC, APC, and APC-Cyanine7 are required. Refer to the user manual for details on hardware requirements for the assay.
See Appendix 4 in the PrimeFlow® RNA Assay User Manual and Protocol for examples of experimental set up. Please contact tech support for help with panel design.