SPOT: an improved differential screening protocol that allows the detection of marginally induced mRNAs.
We have developed a differential screening technique, single plate one transfer (SPOT), that allows the easy detection of mRNAs induced only 2-fold to 3-fold or less above background. As a model system, we looked at the induction of mRNA by parathyroid hormone (PTH) in ROS 17/2.8 rat osteosarcoma cells. The basis for this technique is to symmetrically spot in quadruplicate, on a single plate, a large number of potentially positive plaques obtained from a primary, conventional screen. We then do only one transfer from this plate in order that there will be minimal variability in DNA transfer. This filter is cut into symmetrical strips so that all clones are multiply represented on each strip. These strips are then hybridized with different probes. Since each strip contains an approximately identical amount of DNA per plaque, it is possible to accurately detect mRNAs that are induced only slightly above background. Additionally, the large sizes of the DNA plaques, as well as spotting each clone serially, contribute to the sensitivity of the technique.
Lemke
S J
SJ
Department of Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
Burke
T
T
Boder
G B
GB
Moore
R E
RE
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D015335:Molecular Probes; D010281:Parathyroid Hormone / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D019379:Teriparatide; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D015335:Molecular Probes; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D012333:RNA, Messenger; D019379:Teriparatide; D004247:DNA