TITLE: A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase
AUTHORS: Peter W W Chomczynski, Kianna M Vires, Michal     Rymascewski, Judith   A. Heiny

[DESCRIPTION]
The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the primary α subunit of Na,K-ATPase plays a receptor signaling role in a range of vital cell processes and is a therapeutic target for human disease. Mouse lines with altered affinity for cardiotonic steroids on the α1 or α2 subunit isoform of Na,K-ATPase, without any change in pump activity, were developed by the late Jerry B Lingrel and are a valuable tool for studying its physiological roles and drug actions. In one model, the normally ouabain resistant α1 isoform was rendered sensitive to ouabain binding. In a second model, the normally sensitive α2 isoform was rendered resistant to ouabain binding. Additional useful models are obtained by mating these mice. To further advance their use, we developed a rapid, real-time PCR method that detects mutant alleles using specific primers and fluorescent probes. PCR is performed in fast mode with up to 15 samples processed in 40 min. The method was validated by Sanger sequencing using mice of known genotype, and by comparing results with a previous two-step method that used PCR amplification followed by gel electrophoresis. In addition, we clarified inconsistencies in published sequences, updated numbering to current reference sequences, and confirmed the continued presence of the mutations in the colony. It is expected that a wider availability of these models and a more efficient genotyping protocol will advance studies of the Na,K-ATPase and its cardiotonic steroid receptor.

[STEPS]
SECTION: Pre-experiment preparation
1. In the interest of time and consistency, it is recommended that certain stock solutions and buffers be prepared ahead.
SECTION: Pre-experiment preparation
1.1. Prepare a 10X stock solution of Tail Lysis Buffer and store in freezer.


 

SECTION: Pre-experiment preparation
1.2. 
Prepare assay mixes.
Primers and probes should be pre-mixed and stored frozen for convenience. Be mindful of the reagent stock concentrations to ensure a successful outcome. Mixes can be scaled as necessary. 

 
 
 


 


 


 






SECTION: Pre-experiment preparation
1.3. Prepare Tail Digestion Buffer fresh for each experiment, per the following table. Add proteinase K last from a frozen and thawed aliquot. 

 
 
 
 
 
 
SECTION: Tail clip digestion
2. Clip 2-3 mm from the tail of each mouse to be genotyped. Place in a clean, labelled 1.5 mL microcentrifuge tube.

  


SECTION: Tail clip digestion
3. Add 150 µL of Tail Digestion Buffer containing proteinase K to each tube.

SECTION: Tail clip digestion
4. Place the tail samples in a heat block or water bath. 

SECTION: Tail clip digestion
4.1. Incubate  15 min at 55 °C.
SECTION: Tail clip digestion
4.2. Raise the temperature to 100 °C for 15 min.
SECTION: Tail clip digestion
4.3.  Remove the tubes and allow to cool to 100 Room temperature.
SECTION: Tail clip digestion
5. Briefly vortex and centrifuge the tubes to pellet insoluble material. DNA will remain in the supernatant.

  

SECTION: Tail clip digestion
6. Create a 1:100 dilution of each sample in a new, labelled 1.5 mL microcentrifuge tube. Add 198 µL of TE buffer (or ddH2O) to each tube, then add 2.0 µL of supernatant from the digested sample. 

   

SECTION: Tail clip digestion
7. Store samples and dilutions at 4 °C until ready to genotype. If storing samples longer than 14 days, keep in -20 °C freezer.

SECTION: PCR machine setup
8. Create a new experiment on your PCR machine's software. The instructions here are based on ABI StepOne machines but other manufacturers' procedures are generally similar. 

Templates for ABI StepOne and StepOnePlus machines are attached, which have the probe list and thermocycling program pre-set. To use the templates, download the version corresponding to your machine type. Open StepOne Software and choose [File] -> [New Experiment] -> [From Template...] and select the template file. 

  
  

SECTION: PCR machine setup
9. Set the thermocycling program (run method) as per the following table. 

 

SECTION: PCR machine setup
10. Enter the 4 probes into the targets list as per the following table. Populate the sample list with your mouse sample numbers, plus a no-template control (NTC) sample.

 

SECTION: PCR machine setup
11. Assign 8 wells for each sample (including the NTC); 2 wells for a1R, 2 wells for a1S, 2 wells for a2S, and 2 wells for a2R. For the NTC, mark the wells as negative controls for the respective targets. 

A sample plate layout for genotyping 5 mice is shown here (ABI StepOne). 

 
  


SECTION: PCR reaction setup
12. Prepare 4 reaction mixes as follows, 1 for each of the assay mixes. Create enough mix for the total number of reactions of each assay on your plate map, plus 2 extra to account for potential pipetting loss. 
For example, the plate shown in Step 11 (having 5 samples + 1 NTC) would need a 14 rxn mix for each assay. A full 96-well plate with 11 mouse samples and 1 NTC would require a 26 rxn mix for each assay.

 
  


 
SECTION: PCR reaction setup
13. Pipet 15 µL reaction mix into each well of a fresh PCR plate. 

SECTION: PCR reaction setup
14. Pipet 5 µL of each diluted sample into its assigned wells. Pipet 5 µL ddH2O into each no-template control well.

SECTION: PCR reaction setup
15. Cover the plate with optical sealing film. Ensure that the film is fully adhered to the plate.
SECTION: PCR reaction setup
16. Briefly vortex the plate.
SECTION: PCR reaction setup
17. Centrifuge the plate at 1700 rpm, 2 min .

SECTION: PCR reaction setup
18. Insert the plate into the instrument and initiate the run. 
SECTION: Interpretation
19. Analysis of the PCR results begins by checking the outcome of the negative control wells. If all NTC wells show no amplification, you may proceed with the analysis. If one or more negative control wells show amplification, the results are invalid and the experiment must be repeated. An invalid outcome is typically due to contamination of the reaction mix or of a reagent. 
SECTION: Interpretation
20. Genotyping is based on the presence or absence of signal from the 2 probes of each gene. The example below shows representative amplification plots for the possible genotypes of α1 (A) and α2 (B). The graphs show results from a sample's α1R (black) and α1S (red) wells plotted together (A), and a sample's α2S (black) and α2R (blue) wells plotted together (B). 


   
Samples considered positive for a specific allele show CT values of 26-31 for the corresponding probe, while negative samples do not reach threshold in 35 cycles. Heterozygous samples show amplification of both probes within 2 CT of each-other.