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FlowRNA Assay Frequently Asked Questions (RNA Flow Cytometry)


General Questions

Q. What is the shelf-life of the FlowRNA Assay?

We guarantee the kit performance up to 6 months from the date of receipt.

Q. What cell types are compatible with the FlowRNA Assay?

The assay has been validated for use with primary cells, including human PBMC (cryopreserved, freshly isolated or stimulated), mouse dissociated tissues (such as splenocytes, thymocytes, lymph node cells, and bone marrow cells) and suspension and adherent cell lines. Please contact Tech Support (tech@ebioscience.com) for more information.

Q. Can I use the FlowRNA Assay with whole blood?

Please contact Tech Support (tech@ebioscience.com) for more information.

Q. Are there any precautions I should take with my cells before running this assay?

Yes, we recommend the use of viable, healthy cells that are in the active growth phase. Unhealthy cells are more susceptible to cell lysis during the processing steps in this assay which may result in significant cell loss.

Q. Which species are compatible with the FlowRNA Assay?

We have tested the FlowRNA 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.

Q. Can I use the FlowRNA Assay to analyze multiple RNA transcripts simultaneously?

Yes, the FlowRNA Assay allows for the simultaneous detection of up to three RNA targets.

Q. Is the FlowRNA Assay able to detect RNAi, siRNA or microRNA?

We have optimized the FlowRNA Assay kit to detect cytoplasmic mRNA transcripts, and we do not support RNAi, siRNA or microRNA at this time. Please contact Tech Support (tech@ebioscience.com) for more information regarding other forms of RNA.

Q. To design the probe sets, what is the minimum length of targeted sequence needed?

For optimal sensitivity, a minimum of 1 kb is recommended to design Target Probe sets with sufficient sensitivity. For low expressing genes, a minimum of 2 kb of sequence is recommended.

Q. Do you supply positive control probes as part of the FlowRNA Assay?

Species-specific (mouse and human) positive control probe sets of each of the three types are provided with the PrimeFlow® RNA Assay kit . The Human PrimeFlow® RNA Assay contains type 1, 4, and 6 probe sets for RPL13a, a ubiquitous ribosomal protein. The Mouse PrimeFlow® RNA Assay contains type 1, 4, and 6 probe sets for beta-actin (ACTB). The QuantiGene® FlowRNA (methanol based) Assay does not contain positive control probes. For the QuantiGene® FlowRNA (methanol-based) Assay and for alternative species, positive control genes should be purchased separately. The table below lists additional recommendations for positive controls in other common cells and tissues; however, the appropriate positive control gene for your specific model system should be determined empirically.

Cell

Recommended Positive Control Gene

Human lymphocytes (PBMC) – fresh and cryopreserved

RPL13a, B2M

Human monocytes (PBMC) – fresh and cryopreserved

RPL13a, B2M

Mouse splenocytes (tissue) – fresh and cryopreserved

ACTB, RPL13a

Mouse thymocytes (tissue)

ACTB, RPL13a

Mouse bone marrow

ACTB

Human monocytic lymphoma, U937

RPL13a, B2M

Human T cell lymphoma, Jurkat

RPL13a, B2M

Human cervical carcinoma, HeLa*

RPL13a, GAPDH

Human lung carcinoma, PC9*

RPL13a, GAPDH

Q. Which probe set type do you recommend?

  • Type 1 Alexa Fluor® 647 probe sets provide the most sensitive detection. We recommend Type 1 Alexa Fluor® 647 for target genes with low or unknown levels of expression.
  • Type 4 Alexa Fluor® 488 probe sets are recommended for target genes with medium to high levels of expression. Type 4 Alexa Fluor® 488 is less sensitive than Type 1 Alexa Fluor® 647, in part due to the increase in the autofluorescence of cells caused by the FlowRNA assay protocol .
  • Type 6 Alexa Fluor® 750 probe sets are recommended for target genes with medium to high levels of expression. Type 6 Alexa Fluor® 750 may be preferred to Type 4 Alexa Fluor® 488 if cells are known to have high autofluorescence and greater sensitivity is needed. Detection of the Type 6 Alexa Fluor® 750 probe sets should be in the APC-eFluor® 780 (APC-Cyanine7) channel using a 780/60 bandpass filter, or equivalent, and depends on instrument performance and sensitivity in the infra-red channel. Instrument settings may need to be adjusted as compared to common fluorochrome-conjugated, antibody-based flow cytometric experiments.

Q. Can you detect rare populations in a hetergeneous mix of cells using the FlowRNA Assay?

This assay can be used to detect cell populations that represent greater than 1.0% of the total cells.

Q. How does autofluorescence of the cells affect analysis?

Autofluorescence of cells can reduce resolution between positive and negative events and may make discrimination of dim events difficult. As this will primarily impact Type 4 Alexa Fluor® 488, dim RNA targets should be detected using Type 1 Alexa Fluor® 647 or Type 6 Alexa Fluor® 750 probe sets. Notably, this assay will increase the autofluorescence of cells when compared to live cells or fixed cells. This primarily impacts the FITC, PE, eFluor® 450, and eFluor® 506 channels, but other channels (up to approximately 650 nm) may also be affected, depending on the cell type, laser line, and instrument settings. Please take this into consideration when designing multicolor panels. Please contact Tech Support (tech@ebioscience.com) for more information.

Q. What controls do I need?

  1. To ensure proper assay performance, please use the Positive Control Probe Sets in every experiment. For your convenience, these probe sets are included with the PrimeFlow® RNA Assay kit (RPL13a for human leukocytes and β-actin (ACTB) for mouse tissues). Please refer to Appendix A5 for specific cell types and other recommended genes.
  2. The Positive Control Probe Sets should also be used for single-color compensation controls.
  3. Single-color compensation controls for fluorochrome-conjugated antibodies must also be included in each experiment. We recommend using single-stained cells as compensation controls for antibody staining. However, UltraComp eBeads (cat. 01-2222) may be used. To use UltraComp eBeads, we recommend staining them on Day 1 of the assay, then fixing them with IC Fixation Buffer (cat. 00-8222) so that they may be stored overnight in the dark at 2-8°C. If using a combination of cells and beads, be sure to use the appropriate negative population for setting compensation (for beads, use the negative bead population; for cells, use negative/unlabeled cells that have undergone the PrimeFlow® RNA Assay procedure).
  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.

Q. I am not detecting any fluorescent signal with my probe set. Why is my RNA target not detected?

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 FlowRNA probe sets and that you are using the appropriate compensation controls. Compensation for FlowRNA target probe sets should be conducted using cells labeled individually with positive control probe sets of the same probe type as the experimental targets. 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 FlowRNA 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 (tech@ebioscience.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 FlowRNA Assay due to low expression levels, or inefficiency of unmasking of the mRNA transcript.

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Protocol questions

Q. How many cells should I use per sample for the FlowRNA Assay?

We recommend 1-5 x 106 cells per sample.

Q. I have other microfuge tubes available in the lab, including FACS tubes. Can I use the FlowRNA Assay with other microfuge or FACS tubes?

The use of other microfuge tubes or polystyrene FACS tubes during the hybridization steps of the FlowRNA protocol may result in significant cell loss and weaker signal. The microfuge tubes provided with the FlowRNA 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).

Q. Can the FlowRNA Assay be adapted to a 96-well protocol? Do you have a protocol for this?

We have not validated the FlowRNA Assay for use in 96-well plates. Please contact Tech Support (tech@ebioscience.com) for more information.

Q. I have a 37°C tissue culture incubator in the lab. Can I use this incubator for the hybridization steps?

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 (Affymetrix, cat. QV0523), following the protocol in the FlowRNA user manual. Temperatures deviated outside of this range will lead to less than optimal hybridization.

Q. How critical is it to have the incubator at 40 +/-1°C?

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 recommend the use of [Insert incubator name and cat number here], however any incubator to be used must be validated before use with the ViewRNA Temperature Validation Kit (Affymetrix, cat. QV0523), following the protocol in the FlowRNA user manual. 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 (refer to the user manual for setup details).

Q. I only have fixed-angle centrifuges in the lab. Will this work with the FlowRNA Assay?

The use of a fixed-angle centrifuge may result in cell loss and is not recommended. If a fixed-angle centrifuge must be used, care should be taken during aspiration to avoid disturbing the cell pellet.

Q. I see precipitation in our Target Probe Diluent/PreAmp Mix/AmpMix/Label Probe Diluent. Is this normal?

Yes, visible precipitation in these reagents at colder temperatures is normal. Please pre-warm each of these reagents prior to use, as instructed in the User Manual.

Q. Is the FlowRNA Assay compatible with antibody staining?

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 prior to starting 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. Follow the protocol for antibody staining per the antibody manufacturer’s specifications. 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.

Q. Which fluorochromes are recommended for antibody staining in PrimeFlow® RNA?

Most organic and protein-based fluorochromes are compatible with this assay kit, including PE, PE tandems, APC, APC tandems, and small organic dyes such as FITC, eFluor® 450, eFluor® 660, and Alexa Fluor® 700. BV dyes are also reported to work. However, 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 eVolve™ -antibody conjugates are not compatible with this assay.

Q. Which cytometers are compatible with the PrimeFlow® RNA Assay?

Cytometers with a blue laser (488 nm) and red laser (633-640 nm, or equivalent) and the filter sets indicated below are compatible with the assay. There are three different probe types available, each with its own fluorescence readout.

Probe Set Type

Fluorochrome Label

Excitation Wavelength(max)

Emission Wavelength (max)

Laser Excitation Wavelength

Bandpass Filter Recommendation

Type 1

Alexa Fluor® 647

647 nm

668 nm

632-640 nm

660/20

Type 4

Alexa Fluor® 488

488 nm

519 nm

488 nm

530/30

Type 6

Alexa Fluor® 750

749 nm

775 nm

632-640 nm

780/60

Q. Is this assay compatible with intracellular staining of proteins?

Yes, the fixation and permeabilization buffers for PrimeFlow® RNA Assay is compatible with most cell surface and intracellular (cytokine, transcription factor and some phospho specific) antibody staining with the exception of a few phospho-specific antibodies. Phospho-specific antibody clones that will only work in the IC fix/Methanol protocol are not compatible with this assay kit. Please refer to the Technical Support webpage and the Phospho Flow Cytometry Antibody Clone Buffer Selection Guide or the datasheet for the individual antibody.

The QuantiGene® FlowRNA (methanol based) version is not compatible with intracellular antibody staining. For cell surface markers, please refer to the aforementioned table for the performance of numerous clones and fluorochromes in the methanol-based buffer system.

Q. Do I have to stain with surface staining antibodies before starting the PrimeFlow® RNA Assay?

Staining for some surface markers may be done after fixation and permeabilization. Please see the Antibody Clone Performance Following Fixation/Permeabilization table on the eBioscience website and refer to the column for “After IC Fixation and Perm Wash” to determine if the antibody clone will recognize a fixed epitope.

Q. How would you recommend I set compensation?

We recommend using samples individually labeled with positive control gene probe sets of the same type as your experimental genes to set compensation for each RNA channel. Cells labeled individually with each antibody should be used as compensation controls for those channels. Antibodies with the same fluorochromes as the RNA probes may not be used to set compensation for RNA probes, as the compensation values will be different .Unlabeled cells that have been processed and prepared according to the assay (excluding the target probes) should be used as a negative control for setting compensation.

We recommend using samples individually labeled with positive control gene probe sets of the same type as your experimental genes to set compensation for each RNA channel. Unlabeled cells that have been processed and prepared according to the FlowRNA assay (excluding the target probes) should be used as the negative population for setting compensation. Antibodies with the same fluorochromes as the RNA probes may not be used to set compensation for RNA probes, as the compensation values will be different.

Single-color compensation controls for fluorochrome-conjugated antibodies must also be included in each experiment for each those channels. We recommend using single-stained cells as compensation controls for antibody staining. However, UltraComp eBeads&trade: (cat. 01-2222) may be used. To use UltraComp eBeads, we recommend staining them on Day 1 of the assay, then fixing them with IC Fixation Buffer (cat. 00-8222) so that they may be stored overnight in the dark at 2-8°C. If using a combination of cells and beads, be sure to use the appropriate negative population for setting compensation (for beads, use the negative bead population; for cells, use negative/unlabeled cells that have undergone the PrimeFlow® RNA Assay procedure).

Q. Can we use singly stained antibody samples to set compensation for the different probe types?

Compensation of FlowRNA target probe sets should be conducted using cells labeled with positive control gene probe sets of the same Type as the experimental probe sets. Fluorochrome-conjugated antibodies should not be used for setting compensation for RNA probes.

Q. My cells contain eGFP or another fluorescent protein. Will I be able to detect GFP in cells processed through the assay?

Detection of fluorescent reporter proteins has not been tested and is not recommended. If detection of a reporter protein is required, the use of an antibody targeting this protein during the intracellular antibody staining step may be possible.

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QuantiGene® FlowRNA (methanol based) specific questions

Q. We use paraformaldehyde (PFA) regularly in the lab and have a shared bulk source. How critical is the age and quality of PFA?

We recommend using freshly-prepared, ultrapure paraformaldehyde (PFA), such as the 16% PFA aqueous solution from Electron Microscopy Sciences (EMS, Cat. No. 15710), which is provided as ten, single-use ampules. Use of these ampules ensures that fresh, high-quality PFA is used for each experiment. Using aged or low-purity PFA solution may result in weak to no signal during analysis. This question is not appropriate for PrimeFlow® RNA Assay.

Q. 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.

Q. We store our methanol solution at room temperature. Do we have to cool methanol prior to use with this assay?

Cold methanol pre-chilled at -20°C must be used to ensure optimal fixation and permeabilization of the cells. Sub-optimal permeabilization could result in loss of signal. This question is not appropriate for PrimeFlow® RNA Assay.

Q. Can I use my own buffer and protocol for intracellular protein staining?

We do not recommend changing the buffer and protocol for intracellular protein staining from the FlowRNA protocol. It might alter the sample property and hence affecting the assay performance.

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