Validation of internal standards for expression analysis with real-time PCR in B-CLL.

cDNAs from whole RNA, healthy donor peripheral blood (PB), healthy donor-sorted B cells, and peripheral blood from 3 B-CLL patients were measured with the PE endogenous control plate on an Applied Biosystems 7700 Taqman. The cycle number, when the fluorescence from a PCR reaction reaches a set threshold value, corresponds to the amount of transcript present in the cDNA used as template (Ct value). The average of Ct values from 12 different housekeeping genes was used to normalize all tested patients (norm. Ct values). In contrast to 18S-rRNA andGAPD, PGK, PPI, and LMNB1are expressed at a similar rate in peripheral blood of healthy donors and B-CLL patients and were used as internal standards.