Figure 1
Alu sequence and c-mycERTAM assay development and validation. Gels showing the quality and purity of gDNA by agarose gel electrophoresis (A) and RNA by virtual gel produced by Agilent 2100 Bioanalyzer (B, RNA Integrity Number >9.4 as analyzed by Agilent RNA 600 nano kit [32]) isolated from the same sample. Standard curves used to determine: cell number by Alu sequence qPCR (C, Error 0.0300, efficiency 1.993; 3 replicates); absolute c-mycERTAM copy number by c-mycERTAM qRT-PCR (D, Error 0.0837 and efficiency 2.131; 3 replicates). All standard curves were generated from CTX0E03 gDNA diluted in rat gDNA or cDNA. Crossing point refers to the number of PCR cycles required to generate a detectable fluorescent signal generated on a Roche LC480 instrument. Positive control rat brain samples (B1 and B2) were grafted with approximately 300,000 CTX0E03 cells each and harvested immediately (E, F). Data shown is the total number of CTX0E03 cells in each tissue section as determined by Alu, where control is the number of viable cells in the cell suspension prior to injection as determined by counting using a haemocytometer (E); and total c-mycERTAM transcript copy number calculated per CTX0E03 cell detected in brain samples, where control is the number of copies per cell detected in vitro culture (F).