Myeloid Leukemia

Real-Time RT-PCR Strategies 53
the Fast Technology for Analysis (FTA) card system (Whatman), which is a
chemically treated filter paper designed for storing blood samples for subsequent
DNA and RNA testing (39,40). The FTA cards are impregnated with a
chemical formula that lyses cell membranes and denatures proteins upon contact.
Nucleic acids are physically entrapped, immobilized, and stabilized for
storage at room temperature. Furthermore, cards protect nucleic acids from
nucleases, oxidation, ultraviolet damage, and infectious pathogens. The stability
of genomic DNA on FTA cards for at least 14 yr has been demonstrated.
RNA stability depends on the storage temperature and the type of
biological specimen. RNA of mammalian cells stored on FTA cards is stable
for over 1 yr at temperatures below –20°C and for 2–3 mo in samples stored
at room temperature.
3.4. Normalization and Quantification
3.4.1. Normalization
For normalization, one challenge is to choose one or more endogenous control
gene(s) which take into account factors influencing the different steps of
RQ-PCR. Crucial parameters such as RNA quality and quantity should be
evaluated. This is generally accomplished by amplification, in parallel with the
target gene, of one or more CGs, also called housekeeping or endogenous reference
genes. In the literature, numerous CGs for MRD detection by RQ-PCR are
in use: ABL, BCR, β-actin, GAPDH, PBGD, TBP, and 18S rRNA (Table 2).
A suitable CG in any application of RQ-PCR analysis can be defined as a
gene (1) that is stably expressed in all the nucleated cells among different analyzed
samples and is unaffected by any experimental treatment; (2) that is not
associated with any pseudogenes, in order to avoid genomic DNA amplification;
(3) whose amplification would reflect variations in RNA quality, quantity,
and/or cDNA synthesis efficiency; (4) whose stability should be equivalent
to that of the target gene transcript(s), or whose impaired amplification should
be accompanied by a corresponding reduction in the quantity of target gene
transcript(s); and (5) whose expression should not be very low (Ct > 30) or
very high (Ct < 15).
Generally, one CG is not sufficient for all situations. In order to address the
stability of a given control gene in a series of tissue samples, a robust geneexpression
stability measure was developed by creating an algorithm to determine
the most stable—and hence, reliable—housekeeping genes in a given
tissue panel (41). The algorithm is based on repeated gene stability measurements
and subsequent elimination of the least stable control gene. To handle
the large number of calculations, a visual basic application for Microsoft Excel,
termed Genorm, was developed. Three to five control genes are required for
each tissue (e.g., bone marrow) (41).