PrimeFlow™ RNA Assay Variability

The PrimeFlow™ RNA Assay has been validated for specificity, sensitivity, precision, and accuracy.

Intra-assay Variability

To assess intra-assay variability, samples from stimulated and unstimulated human peripheral blood monnuclear cells (PBMC) were divided into seven tubes and assessed for expression of RPL13a, a positive control gene expressed in all PBMCs, and IFNγ, induced only upon stimulation in a subset of lymphocytes. The target probes were diluted in bulk such that each tube received the same preparation of probes. As shown in the figure below, the assay shows robust intra-assay performance, with CV% of less than 10% for both RPL13a and IFNγ.

Intra-assay Variability

Assessing intra-assay variability
A single sample of stimulated or unstimulated human PBMC was divided into 7 tubes and assessed for expression of RPL13a mRNA in total lymphocytes (figure on the right) or IFNγ mRNA in IFNγγ + events (figure on the left). Data shown are the average of the 7 replicates and error bars represent standard deviation. Values shown above the bars represent the CV%.


To assess the effect of Target Probe dilution on assay variability, a sample of mouse splenocytes was divided into three samples and assayed for expression of β-actin, RPL13a, β2-microglobulin (B2M), GAPDH, and Peptidyl-prolyl cis-trans isomerase B (PPIB). Target Probes were diluted independently for each triplicate. As shown, the CV% were typically less than 10%, while the Type 1 RPL13a Alexa Fluor® 647 probe set was slightly higher, around 12%.

Intra-Experiment Variability by Probe Set

Assessing variability from target probe dilutions
Splenocytes from C57Bl/6 mice were assessed for expression of several positive control genes. Samples were prepared in triplicate with independent dilutions of target probes made for each sample. CV% of the median fluorescence intensity for each gene are shown.

Operator Variability

To assess inter-assay variability, aliquots of U937 cells from the same cell culture were tested in triplicate for GAPDH, PPIB and B2M gene expression by two different technicians. No Probe controls were used as negative controls, and samples were analyzed using the same cytometer settings. For each technician, the CV% of the mean fluorescence intensity (MFI) of triplicate samples was less than 6%, consistent with previous data for intra-assay variability. The variation between technicians was approximately 5% for the MFI, and 20% for the signal to noise ratio, calculated as the ratio between the positive MFI and the No Probe control MFI.

U937 cells were assessed for expression of several positive control genes

Assessing operator variability
U937 cells were assessed for expression of several positive control genes. Samples were prepared in triplicate by two technicians. Histogram overlays of the triplicates for no probe and target probes are shown on the left. MFI for each sample are shown on the right.

Population Variability

Additional studies to assess variation between operators were ran with samples where only a subset of the cells were expected to be positive. C57Bl/6 splenocytes were stimulated for 3 days with anti-CD3 and anti-CD28, then assessed for expression of Ki-67 and Granzyme B mRNA. The samples were then divided among 4 technicians who each independently performed the assay. As shown below, the mean percentage of positive events for Ki-67 or Granzyme B were 18.6% and 30.6%, respectively. The MFI of the mRNA positive events are shown in the bar graph, and in this case the CV was 12-15%, consistent with the results obtained with positive control genes in U937 cells.

Mouse splenocytes were stimulated with Anti-Mouse CD3 (cat. 16-0031) and Anti-Mouse CD28 (cat. 16-0281) Functional Grade Purified antibodies for 3 days, with the addition of brefeldin A and monensin for the last 2 hours.

Assessing operator variability in bimodal expression model
Mouse splenocytes were stimulated with Anti-Mouse CD3 (cat. 16-0031) and Anti-Mouse CD28 (cat. 16-0281) Functional Grade Purified antibodies for 3 days, with the addition of brefeldin A and monensin for the last 2 hours. The cells were stained with Anti-Mouse CD8a PE-eFluor® 610 (cat. 61-0081), Anti-Mouse Ki-67 eFluor® 450 (cat. 48-5698), and Anti-Mouse Granzyme B PE-Cyanine7 (cat. 25-8898), followed by hybridization of Type 4 mouse Ki-67 Alexa Fluor® 488 probe set (cat. VB4-16518) and Type 6 mouse Granzyme B Alexa Fluor® 750 probe set (VB6-16522). Cells in the lymphocyte gate (top) or the mRNA+ events (bottom) were used for analysis.

Day-to-day Variability

To understand day-to-day variation, human PBMC that were stimulated with the Cell Stimulation Cocktail (plus protein transport inhibitor cocktail) (cat. 00-4975) were analyzed fresh or were cryopreserved and analyzed one week later. As shown below, the percentage of positive events is virtually unchanged between the fresh or cryopreserved samples.

Day-to-day variation of the PrimeFlow™ RNA Assaya IFN gamma mRNAb IFN gamma protein
Experiment 1 58.0 90.3
Experiment 2 57.8 87.7
Average 57.9 89.0
Standard Deviation 0.14 1.84
CV (%) 0.24 2.07

aNormal human PBMC were stimulated and analyzed immediately (Experiment 1) or were cryopreserved and analyzed one week later (Experiment 2) for the expression of IFN gamma mRNA and protein. Samples were surface stained with Anti-Human CD8a PE-eFluor® 610 (cat. 61-0088) and followed by hybridization of Type 6 human IFN gamma Alexa Fluor® 750.
bData represent the percentage of IFN gamma+ events, gated on viable CD8+ cells.