Fig. 2.
Validation of the single-cell PCR method. (A) Expression values of cDNA generated from 100 ng total RNA plotted against expression values of cDNA generated and amplified from 50 pg, 10 pg and 1 pg total RNA. Correlation coefficient (R2) values were 0.978, 0.925 and 0.970, respectively. Thus, linearity of relative transcript abundance was well preserved. (B) Addition of exogenous spike RNAs from Arabidopsis thaliana (LTP4, LTP6, NAC1, TIM) allows detection of transcripts of known copy number as an exogenous amplification control (Osawa et al., 2005). The spike RNA samples were diluted to known quantities and added to the first-strand synthesis buffer. Analysing the relative spike cDNA transcript levels after amplification showed that linearity was preserved in samples starting with 50 pg, 10 pg and 1 pg total RNA, where R2 values were 0.994, 0.997 and 0.920, respectively. The spike RNA samples had been diluted to a concentration corresponding to ∼40,000, 4000, 400 and 40 copies, respectively. Detection of 40 copies of a spike RNA demonstrates the sensitivity of the method for amplifying low-abundance transcripts. (C,D) Relative gene expression values of housekeeping genes (ACTB and GAPDH), markers of basal cells (K14, ITGB1) and differentiated cells (K10 and IVL) as measured by QPCR (C) and obtained from Illumina BeadArrays (D). Data show mean ± s.e.m.