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The PrimeFlow RNA Assay is compatible with antibody staining for both surface and intracellular proteins. If surface protein staining is desired, we recommend that you stain cells with antibody according to the instructions under "Day 1: Antibody staining, fixation, and permeabilization" in the assay protocol. Some surface proteins may also be stained at the same time as intracellular proteins, provided that the antibody used recognizes a fixed epitope. Each antibody/fluorochrome must be validated empirically with PrimeFlow RNA Assay to determine its compatibility. A list of validated antibodies for major lineage markers is available online. Please note that antibodies conjugated to PerCP or PerCP tandem dyes are not compatible with the PrimeFlow RNA Assay.

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The green fluorescent protein (GFP) is not compatible with the PrimeFlow RNA assay. In order to detect GFP, we recommend using the Anti-GFP Biotin (Cat. No. 13-6498-80) followed by an appropriate fluorochrome-conjugated streptavidin secondary. Please refer to the Technical Data Sheet (TDS) and the associated Human or Mouse PrimeFlow RNA Assay Protocol for fluorochrome compatibility.

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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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Compensation of PrimeFlow RNA target probe sets should be conducted using the PrimeFlow Compensation Kit included in the PrimeFlow RNA Assay (Cat. No. 88-18005-204, 88-18005-210). Fluorochrome-conjugated antibodies should not be used for setting compensation for RNA probes.

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We have tested the PrimeFlow RNA Assay on mouse and human cells. The assay is expected to work on other mammalian species and has been reported to work in some non-mammalian species. However, this should be determined empirically.

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The PrimeFlow RNA Assay is compatible with whole blood. Please refer to the User Manual for details or contact Tech Support (techsupport@thermofisher.com) for more information.

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Positive control genes, such as RPL13a, beta-actin, beta 2 microglobulin or GAPDH, should be included as a positive control to eliminate operational error and reagent- or equipment-related issues. If signal is observed with the positive control probe set, but not the gene of interest, the following should be considered:

- Ensure that your instrument settings have been optimized for detection of the PrimeFlow probe sets and that you are using the appropriate compensation controls. Compensation for PrimeFlow target probe sets should be conducted using the PrimeFlow Compensation Kit. Fluorochrome-conjugated antibodies should not be used for setting compensation.
- Verify expression of the target gene with other methods of RNA expression analysis such as QuantiGene 2.0 bDNA assay, qPCR, microarray, or RNA sequencing. Ensure that the same sample type, treatment conditions, and time courses are used across the various assays. If gene expression is proven by other assays, verify that the same RNA region is targeted by the PrimeFlow Target Probes. We can design probe sets to cover the same RNA region that has been detected by other assays. Please contact Technical Support for additional assistance (techsupport@thermofisher.com).

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Yes, the PrimeFlow RNA Assay allows for the simultaneous detection of up to four RNA targets.

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The use of other microfuge tubes or polystyrene FACS tubes during the hybridization steps of the PrimeFlow RNA protocol may result in significant cell loss and weaker signal. The microfuge tubes provided with the PrimeFlow RNA Assay have been validated to minimize cell loss and maximize signal. We highly recommend that you only use the provided microfuge tubes. However, cells may be fixed and permeabilized in bulk using polypropylene conical tubes (e.g. Fisher Scientific Cat. No. 14-959-70C). A modified protocol for the use of polystyrene, 96-well plates is also available in Appendix 7 of the PrimeFlow RNA Assay User Manual.

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We have optimized the PrimeFlow RNA Assay kit to detect cytoplasmic mRNA transcripts as well as microRNA. Detection of microRNA is optimal when using the microRNA Pretreatment Buffer and protocol. We do not support RNAi or siRNA at this time. Please contact Tech Support (techsupport@thermofisher.com) for more information regarding other forms of RNA.

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A modified protocol for the use of polystyrene, 96-well plates is also available in Appendix 7 of the PrimeFlow RNA Assay User Manual.

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Positive control genes, such as RPL13a, beta-actin, beta 2 microglobulin or GAPDH, should be included as a positive control to eliminate operational error and reagent- or equipment-related issues. If signal is observed with the positive control probe set, but not the gene of interest, the following should be considered:
- Ensure that your instrument settings have been optimized for detection of the PrimeFlow probe sets and that you are using the appropriate compensation controls. Compensation for PrimeFlow target probe sets should be conducted using the PrimeFlow Compensation Kit. Fluorochrome-conjugated antibodies should not be used for setting compensation.
- Verify expression of the target gene with other methods of RNA expression analysis such as QuantiGene 2.0 bDNA assay, qPCR, microarray, or RNA sequencing. Ensure that the same sample type, treatment conditions, and time courses are used across the various assays. If gene expression is proven by other assays, verify that the same RNA region is targeted by the PrimeFlow Target Probes. We can design probe sets to cover the same RNA region that has been detected by other assays. Please contact Technical Support for additional assistance (techsupport@thermofisher.com).
- Protein expression may not correlate with RNA expression due to differences in expression kinetics and stability of the transcript. For example, changes in the levels of RNA may occur before expression of protein, after which the levels of RNA may drop while the expression of protein remains high. If using an induction model, a time course should be performed to determine the optimal time point to detect expression of the RNA of interest.
- Genes may not be detectable with the PrimeFlow Assay due to low expression levels or inefficiency of unmasking of the mRNA transcript.

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1. To ensure proper assay performance, please use the Positive Control Probe Sets in every experiment. Please refer to Appendix A5 for specific cell types and recommended positive control genes.
2. The PrimeFlow Compensation Kit (included with the PrimeFlow RNA Assay, Cat. No. 88-18005) should be used for single-color compensation controls for the RNA detection channels.
3. Single-color compensation controls for fluorochrome-conjugated antibodies must also be included in each experiment. The UltraComp eBeads microspheres included in the compensation kit may also be used for single-color compensation controls for any experimental antibodies being used in the experiment.
4. In addition to single-color compensation controls, Fluorescence-Minus-One (FMO) controls are highly recommended. The FMO control is a sample that contains all but one of the fluorochromes being used in the experiment. As with single-color controls, there should be an FMO control for every fluorochrome being used in the experiment. FMO controls facilitate assessment of background on gated events and allow fine-tuning of compensation for optimal performance.
5. Negative controls are highly recommended. Samples with the target probe omitted or samples with a target probe not expressed in the cells of interest (such as the bacterial gene DapB) should be used. Additionally, biological or experimental controls comprised of or containing cells known to be negative for the gene of interest (e.g., unstimulated or uninfected) are also recommended to confirm specificity of the target probes.

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We recommend quickly thawing the cells at 37 degrees C according to standard protocols. Wash the cells with culture media to remove DMSO, then allow the cells to rest in culture media at 37 degrees C for 20-30 minutes before proceeding with the PrimeFlow RNA Assay. Do not use benzonase or other nucleases in the thawing medium.

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Proper hybridization is dependent on the sample maintaining a precise temperature. Validation of the incubator is a critical step for success to ensure that the appropriate temperature is maintained. We have available two incubators, Cat. No. QS0704 or QS0712,that have been validated for use with the assay. A metal heat block for 1.5 mL microfuge tubes placed inside the validated incubator is recommended for all hybridization steps to ensure uniform and rapid equilibration to temperature during hybridization. Any incubator to be used must be validated before use with the ViewRNA Temperature Validation Kit (Affymetrix, Cat. No. QV0523), and following the protocol in Appendix 6 of the PrimeFlow RNA user manual.

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