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.
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%.
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.
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.
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|
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.