TempO-Seq DBS
Targeted Transcriptomic Data From A Drop Of Blood
TempO-Seq® DBS for Whole Blood Gene Expression Profiling
BioClavis has developed a novel gene expression profiling tool that enables whole transcriptome monitoring in dried whole blood or blood buffy coat samples spotted on filter paper. Modified chemistry maintains RNA integrity while targeted profiling eliminates unwanted signal from hemoglobin transcripts, dramatically lowering sequencing requirements and costs. Samples taken by a finger prick or collected in a wide variety of standard collection tubes have been used successfully to derive gene expression profiles, as have buffy coat preparations from whole blood, all spotted on filter paper and dried. Dried samples are stable for months, allowing sample collection over time. This assay is compatible with human, rat and mouse TempO-Seq assays, both whole transcriptome and targeted panels, and can be adapted to other species.
Table 1: Evaluated Blood Collection Tubes
Assay Features
Easy Sample Collection
Whole blood samples can be spotted on filter paper directly from a skin puncture using a lancet. Alternatively, blood can be collected by venipuncture and spotted on filter paper. BioClavis has determined that a wide variety of common blood collection tubes are compatible with the TempO-Seq DBS assay, as well as many less common tubes. A list of acceptable blood collection tubes is shown in Table 1. Buffy coat preparations from whole blood can also be spotted on filter paper. Spotted samples should cover up to 2 cm² in area, saturating both the front and the back of the filter paper, and dried at room temperature over several hours. Collection of blood from a finger stick can be facilitated by first warming the hand and finger such as by washing in warm water or using a disposable hand warmer.
Simple Workflow
The TempO-Seq DBS assay maintains the ease of use and progressive addition workflow of the standard TempO-Seq assay. Two 1.6 mm diameter punches are taken into the assay, with an RNase Deactivation Buffer, incubated, and then the standard TempO-Seq assay protocol steps of detector oligo annealing followed by enzymatic digestion of excess unbound detector oligos, ligation of correctly bound detector oligos, amplification of the ligated templates and purification of the sequencing library are carried out. Up to 96 whole blood or buffy coat samples may be assayed in a single run. In addition, sequence results reflect the number of amplified TempO-Seq probes, not donor-specific sequences, thus preventing donor identification based on genetic information.
Assay Performance
Reproducibility
The reproducibility of whole transcriptome data measured by the TempO-Seq DBS assay from finger stick blood was tested by running different replicate regions within the spot for each donor. Figure 2 illustrates the Pearson correlations between all the possible pairwise comparisons between the replicates for each donor sample after log transforming the data. A heatmap of the correlations between finger stick samples collected and tested from the same subject on different days over a period of 400 days is depicted in Figure 3. In this case, all the replicates for the same time point were averaged and the average log transformed values were correlated using the Pearson correlation, with the correlations indicated as the heatmap values
Figure 2: Assay Repeatability
Figure 3: Day-to-Day Repeatability
FIGURE 4: Consistent Differential Expression between Whole Blood and Buffy Coat DBS Input Types
Differentially expressed genes were derived by pairwise comparison of normal donors as measured in whole blood or buffy coat samples. Log2 fold changes between two donors were compared for whole blood vs buffy coat sample input types. Figure 53 illustrates that fold differences in gene expression between the donors correlated well whether profiled from whole blood or buffy coat for all 3 donors.
Summary
TempO-Seq DBS offers a novel way to explore gene expression in blood samples. This technique is simple, robust, and extends the use of the established TempO-Seq assay platform. It can deliver whole transcriptome or focused expression profiles from small amounts of peripheral blood collected using standard methods including a simple finger stick. Spotting and drying samples on filter paper is a key feature of TempO-Seq DBS that enables broad application to blood-based research with minimally invasive sample collection and stable storage. Potential applications include studies of biological responses to experimental or pathological conditions over time in multiple mammalian model systems.