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PrimeFlow™ RNA Assay Frequently Asked Questions (RNA Flow Cytometry)


General Questions

Q. What is the shelf-life of the PrimeFlow RNA Assay?

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

Q. We have the Human (cat. no. 88-18009) and Mouse (cat. no. 88-18001) PrimeFlow RNA Assay.  What is the difference between these catalog numbers and the PrimeFlow RNA Assay (cat. no. 88-18005)?

The Human (cat. no. 88-18009) and Mouse (cat. no. 88-18001) PrimeFlow RNA Assay is capable of simultaneous detection of up to three RNA targets. The PrimeFlow RNA assay (cat. no. 88-18005) enables the simultaneous detection of up to four RNA targets.

Q. Is it possible to add the new channel to the original PrimeFlow RNA Assay (3-plex, cat. no. 88-18001 or 88-18009)?

Unfortunately, no, in order to detect the Type 10 probeset, you will need to purchase the new PrimeFlow RNA assay kit, cat. no. 88-18005.

Q. Is there any change in the protocol with the new channel?

No, there are no changes to the PrimeFlow RNA protocol. The signal amplification components for the Type 10 channel have been incorporated into the corresponding reagents in the kit cat. no. 88-18005.

Q. What is the sensitivity (limit of detection) for RNA staining?

We estimate that under fully optimized conditions, one can detect 10-20 copies per cell for Type 1, Alexa Fluor® 647, or Type 10, Alexa Fluor® 568. The actual sensitivity may vary depending on the specific target.

Q. What cell types are compatible with the PrimeFlow RNA 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 PrimeFlow RNA Assay with whole blood?

The PrimeFlow RNA Assay is compatible with whole blood. Please refer to the User Manual for details or 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 PrimeFlow RNA Assay?

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.

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

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

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

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 (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 PrimeFlow RNA Assay?

Positive control probe sets are sold separately. Please refer to Appendix 5 of the PrimeFlow RNA Assay User Manual or the table below for recommendations and catalog numbers for recommended positive control genes. However, the appropriate positive control gene for your specific model system should be determined empirically.

 

Cell

Recommended positive control gene

Type 1

Type 4

Type 6

Type 10

Human peripheral blood cells*†

RPL13a

VA1-13100

VA4-13187

VA6-13186

VA10-19016

B2M

VA1-10611

VA4-13460

VA6-11782

VA10-10297

U937, human monocytic lymphoma

RPL13a

VA1-13100

VA4-13187

VA6-13186

VA10-19016

B2M

VA1-10611

VA4-13460

VA6-11782

VA10-10297

Jurkat, human T cell lymphoma

RPL13a

VA1-13100

VA4-13187

VA6-13186

VA10-19016

B2M

VA1-10611

VA4-13460

VA6-11782

VA10-10297

HeLa‡, human cervical carcinoma

RPL13a

VA1-13100

VA4-13187

VA6-13186

VA10-19016

GAPDH

VA1-10119

VA4-10641

VA6-10337

VA10-10458

PC9‡, human lung carcinoma

RPL13a

VA1-13100

VA4-13187

VA6-13186

VA10-19016

GAPDH

VA1-10119

VA4-10641

VA6-10337

VA10-10458

Mouse splenocytes*

ACTB

VB1-10350

VB4-10432

VB6-12823

VB10-20653

RPL13a

VB1-16196

VB4-16154

VB6-15315

VB10-20652

Mouse thymocytes

ACTB

VB1-10350

VB4-10432

VB6-12823

VB10-20653

RPL13a

VB1-16196

VB4-16154

VB6-15315

VB10-20652

Mouse bone marrow cells

ACTB

VB1-10350

VB4-10432

VB6-12823

VB10-20653







*Tested on fresh and cryopreserved samples
†PBMC and whole blood; for granulocytes in whole blood samples, B2M is recommended over RPL13a
‡Adherent cells, limited testing

Q. I see four probe set type options. 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 10 Alexa Fluor® 568 is a second sensitive channel that can be used for target genes with low or unknown levels of expression. Detection of Type 10 Alexa Fluor 568 probe sets should be in the PE-eFluor® 610 (PE-Texas Red®) channel using a 610/20 bandpass filter, or equivalent.
  • 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 PrimeFlow RNA 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 heterogeneous mix of cells using the PrimeFlow RNA Assay?

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 10 Alexa Fluor® 568 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. 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.

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 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 (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 PrimeFlow 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 PrimeFlow RNA 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 PrimeFlow RNA Assay with other microfuge or FACS tubes?

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.

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

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

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 Appendix 6 of the PrimeFlow RNA user manual. Temperatures deviating 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 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. QV0523), and following the protocol in Appendix 6 of the PrimeFlow RNA user manual.

Q. I only have fixed-angle centrifuges in the lab. Will this work with the PrimeFlow RNA 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 Wash Buffer/Target Probe Diluent/PreAmp Mix/Amp Mix/Label Probe Diluent. Is this normal?

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

Q. Is the PrimeFlow RNA 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 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.

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® 506, eFluor® 660, and Alexa Fluor® 700. However, PerCP, PerCP-Cyanine5.5, and PerCP-eFluor® 710 may not be used, and we recommend using PE-Cyanine5 or PE-Cyanine5.5 instead. Qdot® nanocrystal and eVolve™ -antibody conjugates are not compatible with this assay.  If Super Bright or Brilliant Violet conjugated antibodies will be utilized, please contact Tech Support (tech@ebioscience.com) for assistance with optimizing your multicolor panel.

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

Cytometers with a blue laser (488 nm), yellow-green (561 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

633-640 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

633-640 nm

780/60

Type 10

Alexa Fluor® 568

568 nm

603 nm

561 nm

610/20

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 most intracellular antibody staining, although some phospho-specific antibodies may be compatible and this should be determined empirically. 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 the PrimeFlow Compensation Kit included in the PrimeFlow RNA Assay (cat. no. 88-18005) 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.

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

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). 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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PrimeFlow microRNA Pretreatment:

Q. Can we detect more than one microRNA in a sample?

Yes, however, due to the limited target probe binding sites on each microRNA molecule, we recommend using Type 1, Alexa Fluor® 647 or Type 10, Alexa Fluor® 568 for maximum sensitivity.

Q. Can we combine microRNA and mRNA staining?

Yes, it is possible to perform any combination of microRNA and mRNA up to a total of 4 targets.

Q. Does the microRNA Pretreatment Buffer affect mRNA staining?

There is no effect of the Pretreatment Buffer on mRNA signal.

Q. Does the microRNA Pretreatment Buffer have any effect on surface staining?

No effects have been observed when surface staining is done before the pretreatment step. If staining for surface markers is done after fixation & permeabilization, the addition of the pretreatment step may have some adverse effects for some clones. Antibody performance after pretreatment, fixation, and permeabilization should be determined empirically.

Q. In the table for performance of intracellular antibodies, does this mean all formats of the clones listed with a negative are not compatible with the PrimeFlow RNA Assay?

In the case of antibodies for Foxp3, we believe the clones do not stain after the pretreatment step, so we do not expect any formats for those clones to work.

Q. How does the data look for microRNA staining if we use the standard PrimeFlow RNA Assay protocol?

In some cases, there is a decrease in the signal for a given microRNA target, in other cases there is no difference. The extent of the decrease and whether or not it occurs varies depending on the specific microRNA target being analyzed.

Q. Are tandem dyes affected by the microRNA Pretreatment Buffer?

No changes for tandem dyes have been observed with the Pretreatment Buffer.

Q. Will there be an expiration date on the bottle?  If not, what is the shelf-life?

Expiration dates are printed on the labels of each component.

Is the microRNA Pretreament Kit compatible with the original PrimeFlow RNA Assay (3-plex)?

Yes.

Q. Is the microRNA Pretreatment kit required for microRNA detection in the PrimeFlow RNA Assay?

The Pretreatment Buffer is not absolutely required for microRNA detection. However, it can enhance detection for most of the microRNA targets tested.

Q. What is the sensitivity for microRNA detection?

The sensitivity is estimated to be 40 copies per cell with Type 1, Alexa Fluor® 647 Probe Sets. The actual sensitivity may vary depending on the specific target and Probe Type.

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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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96-well PrimeFlow RNA protocol

Q. 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.

Q. What is the recommended method for removing supernatant after each centrifugation?

To discard supernatant from the wells, the plate may be inverted, using a single motion with adequate force ("flicking"), and then gently blotted on a paper towel. Alternatively, aspiration may be used, being careful to not disrupt the pellet. The residual volume inside each well should not exceed 10 µL.

Q. I have spun the plate and cannot see my cell pellet. Is this normal?

Depending on the starting cell number and the cell type used, the cell pellet may not be easily visible. For 2 million cells of PBMC, an opaque cell pellet is barely visible in a v-bottom plate.

Q. What type of 96-well plate is compatible to the protocol?

We recommend polystyrene uncoated v-bottom 96-well plates with a lid (cat. # 44-17005). The u-bottom plate is also acceptable, but flat bottom plates are not recommended.

Q. Will the use of 96-well plates increase the amount of cell loss compared to the tube protocol? What type of cell loss should I expect?

The cell loss may be slightly higher in 96-well plates compared to the tube, depending on the method used to remove supernatant after washes (manually or using an aspirator), but should be less than 50%.

Q. If I am running multiple plates, can I stack plates in the incubator during any of the hybridization steps?

This protocol is optimized for analysis of 1-2 plates. If using multiple plates, place each plate directly on the shelf of the incubator; do not stack plates during hybridization

Q. Is there an edge effect seen with the 96-well plate protocol?

The variation between wells, whether on the edge or in the interior of the plate, is similar to the variation observed between tubes and is within the assay specifications.. However, if only partial portion of the plate is needed, we recommend placing the samples in the center of the plate.

Q. At the end of the protocol, there is a note that the samples need to be transferred to a flow tube. I have a 96-well adaptor on the flow cytometer. Can I use the adaptor to acquire the data from the plate directly?

Yes, the samples can be collected directly from the plate with a 96-well adaptor

Q. During the hybridization steps in the incubator, do I need to cover or seal the plate with a plate sealer to prevent condensation and evaporation?

It is necessary to keep the lid on the plate during hybridization, but sealing is not required.

Q. If the plates are stored overnight at 4oC after Day 1 procedures, should we spin the plates first before continuing with the PrimeFlow RNA assay?

Normally, spinning is not required. If there is significant condensation after overnight storage, spin plate at 400 xg for 1 minute before proceeding..

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