When running the QuantiGene FlowRNA (methanol based) assay, which fluorochromes are recommended for antibody staining?
We recommend the use of fluorochromes that are known to be methanol resistant. Avoid protein-based fluorochromes such as APC, PE, and PerCP and their respective tandems. This question is not appropriate for PrimeFlow RNA Assay.
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For the PrimeFlow RNA Assay, how would you recommend I set compensation?
We recommend using the PrimeFlow Compensation Kit included in the PrimeFlow RNA Assay (Cat. No. 88-18005-204, 88-18005-210) to set compensation for each RNA channel. Refer to Appendix 3 of the PrimeFlow RNA Assay User Manual for detailed instructions for use. The UltraComp eBeads microspheres included in the compensation kit may also be used for any experimental antibodies being used. Antibodies with the same fluorochromes as the RNA probes should not be used to set compensation for RNA probes, as the compensation values will be different.
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I have a 37 degrees C tissue culture incubator in the lab. Can I use this incubator for the hybridization steps in the PrimeFlow RNA Assay protocol?
The hybridization temperature is a critical parameter of this protocol necessary to obtain positive results. The incubator to be used must be validated before use with the ViewRNA Temperature Validation Kit (Cat. No. QV0523), following the protocol in the FlowRNA user manual. Temperatures deviated outside of this range will lead to less-than-optimal hybridization.
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What type of 96-well plate is compatible with the PrimeFlow RNA protocol?
We recommend polystyrene uncoated v-bottom 96-well plates with a lid (Cat. No. 44-17005-46). The u-bottom plate is also acceptable, but flat bottom plates are not recommended.
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How many cells should I use per well for the 96-well format of the PrimeFlow RNA Assay?
We recommend 1-5 million cells in 100 µL of Flow Cytometry Staining Buffer per well. The starting cell number is critical for maximal signal intensity.
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