The overall goal of this protocol is to quantify the proportions and absolute levels of STAT3 splice variant transcripts in eosinophils. This method can address key questions in the splicing regulation field such as coassociation of distal splicing events and identification of conditions that can cause tandem splicing proportions to differ. The main advantage of this technique is the plasmid standards enabling acquisition of absolute and relative qPCR data so we can determine proportions and overall levels of four STAT3 transcripts.
In our case we wish to learn how the four STAT3 variants S alpha, S beta, delta S alpha, and delta S beta change when eosinophils are stimulated by cytokines. Though this method can provide insight into eosinophil STAT3 composition it can also be applied to other cell types for which cDNA can be obtained. Because S and delta S vary in only three nucleotides we had to sacrifice efficiency to achieve specificity in either standards standards for each variant.
In this protocol cDNA prepared from human eosinophils is used as template for amplifying the STAT3 splice variants. After creating the plasmids as described in the text protocol prepare sequencing reactions for sequencing plasmids from several colonies. Each 20-microliter reaction should contain three microliters of sequencing reaction buffer, two microliters of sequencing reaction mix, 12 microliters of ultrapure water, one microliter of plasmid, and two microliters of sequencing primer.
When the sequencing results are available assess the electrophoretograms of the plasmids encoding each variant;S alpha, S beta, delta S alpha, and delta S beta. Measure the concentration of pure plasmid DNA in nanograms per microliter and calculate the copy number per microliter. Begin this procedure by preparing a one in 10 dilution series of the STAT3 plasmids using ultrapure water.
With approximately 10 to the eighth to 10 to the third copies per microliter. Measure the concentration of the most concentrated sample. Use the diluted plasmids to prepare four nontarget mixes as negative controls for each splice variant at 10 to the sixth copies per microliter for each variant.
Prepare a target mix with equal concentrations of all four templates, each at 10 to the sixth copies per microliter. Prepare primer pair solutions for each splice variant with 60 microliters of primers, each at a seven micromolar concentration, for a final concentration of 560 nanomolar in the sample. Dilute the DNA polymerase, double-stranded DNA binding dye mix, seven to five with pure water.
Set up the assay in a 96-well PCR plate following this template. First add to each well 21 microliters of the diluted DNA polymerase, double-stranded DNA binding dye mix. Then add two microliters of primer mix and two microliters of template.
To each no-template control well add two microliters of filtered water instead of template. Set up the reactions in duplicate to assess repeatability. Seal the qPCR plate with an adhesive cover and centrifuge for five minutes at 1, 200 g at 12 degrees Celsius.
Turn on the qPCR machine and insert the sealed qPCR plate. If using the ABI real-time PCR system, set up the experiment as demonstrated. Click File, New, Next, select new Detector, then provide a name and choose a Reporter and a Quencher.
Set up a new detector for each splice variant. Select Next and set up the standards as prompted on the Set Up Sample Plate page ensuring the quantities are entered into the table and the appropriate detectors are selected. Click Finish.
Set up the appropriate PCR cycling program. Ensure that the fluorescence reading to determine threshold cycle values occurs during the 72 degrees Celsius step of each cycle. Select Run.
When the run is complete click the Results tab Amplification Plot tab. Evaluate the Output Amplification plot to assess the data quality. An exponential plot indicates reliable amplification.
Export the results to a spreadsheet software. Analysis of the data as described in the text protocol is key to knowing whether the assay requires further optimization for the sake of reproducibility. The cDNA for this assay is prepared from eosinophils treated with cytokines to promote transcription.
Prepare serial dilutions of two sample cDNA aliquots with a dilution range from one to one in 1, 024. Set up the assay in a 96-well plate with 25-microliter reactions as demonstrated for analyzing primer specificity but with several primer concentrations for pan STAT3 and a housekeeping gene GUSB. A template is shown in this table.
Insert the sealed PCR plate into the qPCR machine. Set up the experiment using the software adding a new detector for GUSB and using the appropriate PCR cycling program. Subsequently, the results are used to calculate the threshold cycle value for each sample as described in the text protocol.
To begin this procedure compare the threshold cycle values obtained in relative qPCR for the housekeeping gene GUSB and dilute cDNA accordingly with ultrapure water to ensure all concentrations are within one order of magnitude. Set up the absolute qPCR assays of the samples in 96-well plates. Follow this template for S alpha and S beta and this template for delta S alpha and delta S beta.
Carry out the assays as shown previously and repeat the assays at least once. Next, set up an absolute qPCR 96-well plate with 25 microliter reactions using pan STAT3 primers at 400 micromolar and a mix of all four plasmids or a single plasmid following this template. Seal the qPCR plate with an adhesive cover and centrifuge for five minutes at 1, 200 g at 12 degrees Celsius.
Insert the sealed PCR plate into the qPCR machine and set up the experiment as shown previously adding a new detector for pan STAT3. Set up the same cycling parameters as for relative qPCR. After repeating the assay at least once analyze the results as described in the text protocol.
Good quality qPCR data will generate a sigmoidal amplication plot signifying exponential increase in transcripts over the course of cycling. These data were obtained from qPCR of two serial dilutions of plasmid containing STAT3 S alpha. Each pair of colored lines represents the fluorescence levels of duplicate diluted samples over the course of 40 cycles.
Standard curves generated for the template calibrator plasmids will indicate the efficiency of amplification. In this curve for STAT3 S alpha the amplification efficiency was 83.9%In order to evaluate the congruency of STAT3 levels the absolute values of each of the four splice variants were measured as well as the level of total STAT3. Ideally, both the linear regression and slope should be close to one.
The absolute qPCR data are presented as pie charts to show their proportions of the four STAT3 splice variants over the course of cytokine treatment. Merging absolute and relative qPCR data demonstrated that levels of all STAT3 splice variants increased post stimulation with cytokines IL3 and TNF alpha with levels peaking at six hours. Pooling the data from all time points reveled a small but significant bias for coassociation of the beta and delta S splicing events.
Once optimized, assays based on this technique can be performed in four hours if performed properly. While attempting this procedure it's important to remember to work under sterile conditions to prevent contamination. Splicing decisions that include or exclude one codon account for 20%of in-frame splice variation.
Our protocol should be readily adaptable to quantification of such variations. After its development, this technique allowed Mao Zhang to work with my group in the group of Li-Shan Rue to control knockdown re-expression experiments of STAT3 variants in ABC lymphoma cells. These experiments demonstrated that a mix of S and delta S variants are required for cell survival in culture.
After watching this video you should have a good understanding of how to perform absolute and relative qPCR and the conditions appropriate for distinguishing and quantifying subtle splicing differences.
