Nancy E. Cook

Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology


RNA was extracted and purified from immature soybeans seeds at the R4 and RS stages. Riboprobes for the sense and antisense sequence of a CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (CPT) cDNA were transcribed and labeled with biotin. RNA's from the gene coding for CPT were hybridized to the biotin-labeled riboprobe during ribonuclease protection assays (RP A). The RP A: Ribonuclease Protection Assay Kit (Arnbion) protocol and reagents were used to combine measured quantities of RNA and biotin-labeled riboprobe. After incubation, the protected fragments were extracted and resuspended in gel loading buffer. Samples from the RPA's were separated in thin polyacrylamide gels and transferred onto nylon membrane with an S&S TurboBlotter. Biotin-labeled probe was detected using streptavidin-alkaline phosphatase (SAAP) and Lumi-Phos 53QT'-' (United States Biochemical) and exposing the membrane to x-ray film. The film was developed, scanned, and saved as a TIFF file using the Ofoto program. Blots were quantified with the NIH Image program. Relative intensities were statistically compared using the GraphPad InStat Mac program. The data show no significant difference between the quantified RNA for three developmental stages.

Library of Congress Subject Headings

Phosphotransferases Cytidine diphosphate choline Soybean -- Seeds



Number of Pages



South Dakota State University