Figure 10. Verification of quantitative accuracy.

(A) Two cDNA targets were quantified using LRE (N₀  =  transcripts per 10 ng of total RNA), based on four replicate amplification reactions. The tight clustering of the replicate profiles is reflective of the high level of precision generated by these amplicons. Note, however, that for unknown reasons RNase H treatment has been found to dramatically disrupt replicate profile clustering for some amplicons (data not shown). (B) Samples were diluted to a predicted target concentration of about 0.5 N per aliquot and 16 replicate aliquots amplified. Similar to Figure 8B, a high level of profile clustering was generated, including putative 1 N and 2 N clusters for the 46630 target. The LDA quantifications, which rely solely on the frequency of nil reactions, correlate well with the LRE-based quantifications. (C) Counting target molecules as illustrated in Figure 8B correlates well with both LRE and LDA quantification, supporting the contention that these clusters were produced by amplification of one or two target molecules. (D) Summaries of the LRE analysis of the 1 N and 2 N profiles further demonstrate the ability of LRE to maintain quantitative accuracy down to a single target molecule. Note also the precise one cycle separation between the putative 1 N and 2 N clusters from the 46630 target, which is consistent with a one-fold difference in target quantity.