Search
Search
View additional product information for PrimeFlow™ RNA Assay Kit - FAQs (88-18005-204, 88-18005-210)
51 product FAQs found
The Brilliant Violet dyes are reported to work with the PrimeFlow RNA Assay kit. For more information on fluorochrome compatibility, please check the following link.
Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center
Positive control probe sets are sold separately. Please refer to Appendix 5 of the PrimeFlow RNA Assay User Manual 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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The Pretreatment Buffer is not absolutely required for microRNA detection. However, it can enhance detection for most of the microRNA targets tested.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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 at techsupport@thermofisher.com for assistance with optimizing your multicolor panel.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Staining for some surface markers may be done after fixation and permeabilization. Please see the Antibody Clone Performance Following Fixation/Permeabilization table on the website (www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/cell-analysis-resource-library/ebioscience-resources/antibody-fixation-considerations.html) and refer to the column for “After IC Fixation and Perm Wash” to determine if the antibody clone will recognize a fixed epitope.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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 email Technical Support or refer to the 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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
This assay can be used to detect cell populations that represent greater than 1.0% of the total cells.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Yes, the PrimeFlow RNA Assay allows for the simultaneous detection of up to four RNA targets.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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 (techsupport@thermofisher.com) for more information.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Normally, spinning is not required. If there is significant condensation after overnight storage, spin plate at 400 xg for 1 minute before proceeding.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
It is necessary to keep the lid on the plate during hybridization, but sealing is not required.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Yes, the samples can be collected directly from the plate with a 96-well adaptor.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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%.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Cold methanol pre-chilled at -20 degrees 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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
A modified protocol for the use of polystyrene, 96-well plates is also available in Appendix 7 of the PrimeFlow RNA Assay User Manual.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
We recommend 1-5 x 10e6 cells per sample.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
For optimal results, we recommend that you analyze stained cells immediately.
However, if you do need to store your samples, we recommend you complete the PrimeFlow RNA protocol and fix your samples with IC Fixation Buffer (Cat. No. 00-8222-49, 00-8222-52) (100 µL sample with 100 µL IC Fixation Buffer). Cells can be stored in these buffers for up to 3 days in the dark at 4 degrees C.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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 (techsupport@thermofisher.com) for more information.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
We guarantee the kit performance up to 6 months from the date of receipt.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.