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Automating RNA Isolation

esults from the analysis of 9 samples from K562 cells are shown in Figure 2. Cycle threshold (Ct) values, which reflect the amount of target RNA present in the sample, were calculated (Figure 3). Although some variation in yield between replicate wells was observed, it is most likely due to several factors unrelated to the isolation method, including variation in the number of cells manually dispensed to each well, and variation in cell growth and turnover during incubation. Importantly, the consistent ratio of hTERT to GAPDH in replicate samples indicates that both high abundance and low abundance mRNAs are reproducibly isolated with the automated RNAqueous-96 protocol.

Figure 2.Real-time RT-PCR Detection of GAPDH and hTERT in Replicate Samples Isolated Using the RNAqueous-96 Automation Protocol. RNA was isolated as described in Figure 1. Samples were analyzed by real-time RT-PCR using primers and a TaqMan probe for GAPDH (A.) or hTERT (B.) in an ABI 7700 Sequence Detector. Data from 9 samples, each indicated by a different color line, are shown here. The average cycle threshold (Ct) values for all of the samples are shown in Figure 3.


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Cell Line Target Average Ct Value Standard Deviation
K562 GAPDH 15.86 0.45
hTERT
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