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the RNA of PBMCs over time, we analyzed total RNA obtained from 8 individuals at t 3 , t 5 , t 10 , and t 15 of PBMC storage time with reference to their t 0 values. For all samples examined, RNA purity, determined by the A 260 / A 280 ratio, was 1.8 –2.1, indicating that highly pure RNA could be obtained with storage in buffer E. In addition, for all 3 genes, we observed no significant variations in either the amount (Fig. 1C; Friedman test, P ϭ 0.0812) or the integrity (Fig. 1C of the online Data Supplement; Fried-man test, P ϭ 0.3682) of RNA at t 3 , t 5 , t 10 , and t 15 compared with t 0. Thus, buffer E provides advantageous conditions for PBMC storage at Ϫ80 °C. In addition, it is noteworthy that buffer E is also suitable for manual extraction (see the manufacturer's instructions), which does not necessarily require specific and costly automation. The kinetics of gene expression and RNA stability in blood samples processed ex vivo (preanalytical variables) are currently under intense investigation (6, 12, 13). To guarantee optimal conditions, samples reach our laboratory for processing after a transportation time of Ͻ2 h at 4 °C. Indeed, gene expression was shown to change 2 h after blood collection (13), probably because of significant stress responses (12) or possible contact activation of cells in the collection tubes (9). These findings underscore the extreme sensitivity of blood cells to ex vivo handling. One study has suggested that EGTA is preferable to EDTA as an anticoagulant to prevent the irreversible loss of anti-gen-specific lymphoproliferative responses in instances in which unclotted blood may be stored for long periods (14). Methods such as the PAXgene tube system or addition of acidic phenol and guanidine isothiocyanate have been proposed to stabilize blood cell gene expression after prolonged incubation of blood ex vivo, but new methods and protocols are needed (8, 12). Our studies demonstrate that PBMC RNA is stable over time (up to 15 months) when blood samples are collected into EDTA tubes and when PBMCs are stored at Ϫ80 °C in buffer E. We provide here a simple assay for rapid, efficient, and standardized banking of PBMCs, a key step in large gene expression studies. To our knowledge, this is the first comprehensive report on RNA stability over 15 months. Moreover, preliminary observations …

Denaturing HPLC Coupled with Multiplex PCR for Rapid Detection of Large Deletions in Duchenne Muscular Dystrophy Carriers