Growth conditions

Protocol for diagnostic qPCR for aneuploidy

This protocol was designed to check aneuploidy status at various points before, during, and after experiments.  The procedure measures the relative abundance of each amplified chromosome (Chr12 in this case) to genes on a reference chromosome that is not duplicated.

1. Aneuploid (and control euploid) cells were collected. 
2. Genomic DNA (“gDNA”) was prepped with Zymo Research's YeaStar Genomic DNA Kit (Cat # D2002). Optimal zymolyase digestion was achieved with 1mL cells of OD600 of 1 or less. gDNA was quantified with a Nanodrop 2000. 
3. 1ng gDNA with final primer concentrations of 150nM was used in a 20uL reaction with Roche's LightCycler 480 SYBR Green I Master (Cat # 04 707 516 001) as outlined in the SYBR Green I Master manual. 
   Primer note: For Chr12, select genes were chosen (SDH2, AAT2 and SKI2) and compared against non-Chr12 genes, encoded on other chromosomes (ACT1 or ERV25). Because Chr12 is a large chromosome, we chose multiple genes located on each arm, including the far end of the larger right arm, to ensure that the entire chromosome remained duplicated. 
4. The delta-delta-Ct method was used for quantification, first normalizing the abundance of each Chr12 gene to the non-Chr12 control gene in that sample, and then subsequently comparing that relative value in each aneuploid strain to a control euploid strain. To do this, Ct values were obtained from the instrument for each strain/primer combination (done in technical triplicate). Chr12 gene Ct values were subtracted from the control-gene Ct value (since Ct reflects abundance in log₂ space), and then this relative difference was compared between aneuploid and euploid strains.  Values were converted to linear space.
5. Relative delta-delta-Ct values were interpreted as follows: Aneuploid-euploid delta-delta-Ct values of technical-average 2.0 (1.0 in log2 space) were expected for maintenance of Chr12 disomy in haploid strains, where as a technical-average value of 1.0 (0 in log2 space) was expected for complete reversion to euploidy.  Values in the range of 1.2-1.8 were taken to reflect a partial loss of aneuploidy in the cell population (i.e. mixed aneuploid-euploid culture) at the end of the experiment.  

Primers used: 

        SDH2 Fq 5’-CGTGCTAATGCAAGCCTACCG-3’

        SDH2 Rq 5’-ACCAGGATTCAAGCCCTTTGGAC-3’

        AAT2 Fq 5’-ATGTCCTCCAGAATTACGAAAATGA-3’

        AAT2 Rq 5’-TCAATCCAGCAATAGAAGCTCTACC-3’

        SKI2 Fq 5’-CATTAACTCAGGATGAAGCTG-3’

        SKI2 Rq 5’-CGTCTAGCCAAGTGATAACTC-3’

        ERV25 Fq 5’-GGAACAAGATCAGTGCCAACG-3’

        ERV25 Rq 5’-TGACTTGCCAAACACCCAGC-3’

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