Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (ΔS) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (α) or a truncated transactivation domain with 7 unique residues (β). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (Sα, Sβ, ΔSα and ΔSβ) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites

Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells.

Signal transducer and activator of transcription 3 (STAT3) is a key mediator of leukocyte differentiation and proliferation. The 3' end of STAT3 transcripts is subject to two alternative splicing events. One results in either full-length STAT3α or in STAT3β, which lacks part of the C-terminal transactivation domain. The other is at a tandem donor (5') splice site and results in the codon for Ser-701 being included (S) or excluded (ΔS). Despite the proximity of Ser-701 to the site of activating phosphorylation at Tyr-705, ΔS/S splicing has barely been studied. Sequencing of cDNA from purified eosinophils revealed the presence of four transcripts (S-α, ΔS-α, S-β, and ΔS-β) rather than the three reported in publically available databases from which ΔS-β is missing. To gain insight into regulation of the two alternative splicing events, we developed a quantitative(q) PCR protocol to compare transcript ratios in eosinophils in which STAT3 is upregulated by cytokines, activated B cell diffuse large B cell Lymphoma (DLBCL) cells in which STAT3 is dysregulated, and in germinal center B cell-like DLBCL cells in which it is not. With the exception of one line of activated B cell DLCBL cells, the four variants were found in roughly the same ratios despite differences in total levels of STAT3 transcripts. S-α was the most abundant, followed by S-β. ΔS-α and ΔS-β together comprised 15.6 ± 4.0 % (mean ± SD, n = 21) of the total. The percentage of STAT3β variants that were ΔS was 1.5-fold greater than of STAT3α variants that were ΔS. Inspection of Illumina's "BodyMap" RNA-Seq database revealed that the ΔS variant accounts for 10-26 % of STAT3 transcripts across 16 human tissues, with less variation than three other genes with the identical tandem donor splice site sequence. Thus, it seems likely that all cells contain the S-α, ΔS-α, S-β, and ΔS-β variants of STAT3

<i>STAT3</i> S-β and ΔS-β splice variant transcripts are present in eosinophils.

<p>PCR products were cloned into pET-Elmer vectors, amplified in <i>E</i>. <i>coli</i> and sequenced. The electrophoretogram depicts sequencing data from PCR amplification with a reverse primer specific for <i>STAT3β</i>. The amino acids encoded by the complementary sequence are shown below.</p

Validation of primer specificity.

<p>The specificity of primers uniquely amplifying the four <i>STAT3</i> splice variants was validated using mixed isoforms as template in PCR. An annealing temperature of 66°C was required to exclude S-α and S-β amplifications from the ΔS-α and ΔS-β reactions, respectively. DNA ladder is exACTGene 1 Kb Plus DNA Ladder (Fisher Scientific International Inc., Waltham, MA).</p

Primers used for quantitative PCR analysis.

<p>Primers used for quantitative PCR analysis.</p

Pan-<i>STAT3</i> primer quantification was compared to the summation of the four splice variants.

<p>Quantification was performed for 18 eosinophil or DLBCL samples to validate the absolute qPCR protocol. Each point is plotted with its CV and represents the average of three replicates.</p

Splice variant ratios of eosinophil <i>STAT3</i> change little when <i>STAT3</i> transcription is induced.

<p><b>(A)</b> Relative quantification qPCR was used to determine fold changes in overall <i>STAT3</i> levels in eosinophils over time under different conditions determined using <i>GUSB</i> as a reference gene. <b>(B)</b> Pie charts of <i>STAT3</i> splice variants in untreated and treated eosinophils. Absolute quantification qPCR was used to determine proportions of <i>STAT3</i> splice variants. Percentages of each splice variant are shown. Data represent averages determined from at least three reactions, with standard deviations shown for (A). IL3 = interleukin 3. TNFα = tumor necrosis factor α.</p