John M. S. Bartlett.pdf - Bio-Nica.info

AU-Differential Display 233
Fig. 4. Confirmation of the differential expression by specific RT-PCR (primers derived from
sequences of cloned AU-DD products) in agarose gels of a few AU-DD fragments taken as
examples. Odd lanes are from activated cells and even lanes are the counterpart resting cells.
the system sensitivity and reproducibility on the IL-2 cDNA. However, when used to
supplement standard primers, a small but consistent gain in the number of products
obtained from total cDNA was noticed, even when sensitivity to the IL-2 standard was
not improved (not shown). Profile reproducibility among replicas was excellent for the
primers and conditions described.
5. Notes
1. Different RNA preparation methods were examined in function of copurifying RNase
activities, contaminating DNA, and yield. Ultracentrifugation in Cs salts (CsCl, CsTFA)
gave an acceptable low level of DNA, but it was discarded because of its relatively
poor yield. Phase lock heavy gel was found to improve both the yield in RNA and the
partitioning of the genomic DNA to the lower phenolic phase. The single step acid-phenol
method (14) was used with minor modifications intended to ensure RNA integrity and
an efficient removal of both RNase activities and of contaminating genomic DNA. The
procedure is described since this latter factor is considered relevant to the main method.
2. Fresh tissue is snap frozen in liquid nitrogen and then stored at –80°C or lower until
needed. Ceramic mortar is precooled by pouring on it liquid nitrogen where pestle is
submerged; it is advised to wear gloves during this procedure. Initial bubbling will cease
when the tool is cool enough. Then, the tissue is submerged and ground till reduced to
a powdered state. The remaining liquid nitrogen is left to evaporate until the tissue is
just wet, like a paste. Then, it is poured to a polypropylene 50-mL conical centrifuge
tube. As soon as the tissue starts looking dry, add the lysis solution, mix by vortexing
and homogenize.