Myeloid Leukemia

122 Rossi, Levati, and Biondi
Fig. 2. Ethidium bromide-stained agarose gel showing instability of PML-RARA
transcripts. The gel shows bcr3 +ve nested polymerase chain reaction (PCR) products.
(A) 1 = positive control, 2 = BM sample of patient at diagnosis, 3 = follow-up bone
marrow (BM) sample that has been processed immediately, 4 = the same follow-up
BM sample processed after 24 h and analyzed after 48 h, 5 = negative control (water
only, no cDNA template), MK = marker. (B) Amplification of Abl-1 control gene for
the same samples.
ever, that approach can cause rapid degradation of RNA during the process of
freezing and thawing. In our experience, even the immediate lysis of BM samples
without Ficoll density separation does not result in RNA samples of good quality
for further analysis. Samples that cannot be processed immediately should be
kept at 4°C, but not frozen, for not longer than 24 h. If the sample is delivered to
a referral laboratory, it should be sent at 4°C. Ficoll density separation should be
performed immediately upon receipt of the BM sample, and in any case no later
than 24 h following sample collection. The use of nucleic acid preservative
reagents has been shown to be useful for increasing RNA stability in PB samples
(16), but less effective in BM, and accordingly is not applicable to APL samples.
4. Integrity of RNA. An aliquot of RNA is run on 1% agarose gel electrophoresis,
and a partial assessment of RNA integrity is based on the presence and quality of
RNA ribosomal bands.
5. If necessary, increase the amount of RNA to be retro-transcribed in vitro (2–5 µg
instead of the routinely used 1 µg).