Changes in the Temporal and Spatial Expression: MATERIALS AND METHODS(5)

13 Jun
2013

MATERIALS AND METHODS(5)

Ribonuclease Protection Assays

The rat Hp58 cDNA that was isolated and cloned from the RNA fingerprinting analysis was restricted with HindIII and transcribed in vitro using T3 polymerase to produce single-stranded RNA probes using the Riboprobe kit (Pro-mega Biotech, Madison, WI) and [a-32P]CTP (800 Ci/ mmol; ICN). To adjust for assay variability a rat glyceral-dehyde 3-phosphate dehydrogenase (G3PDH) riboprobe (Ambion, Inc., Austin, TX) was synthesized as a control mRNA using SP6 polymerase. The transcripts were gel purified, eluted from the gel overnight, and added to the RNA samples. Total placental or labyrinthine cell line RNA (10 ^g) was combined with the single-stranded probes (—20 000 cpm, Hp58 and ~1000 cpm, G3PDH), and reactions were adjusted to contain 50 ^g total RNA using yeast RNA. Ribonuclease protection assays were performed using the HybSpeed RPA kits (Ambion) as described by us previously. Briefly, samples were hybridized according to the manufacturer’s protocol, and free riboprobe was removed by digestion with a 1:100 dilution of a mixture of RNase A (500 units/ml) and RNase T1 (20 000 units/ml) for 30 min at 37°C. Protected fragments were separated on polyacrylamide (5% w/v) urea (8 M) gels. The RNA size markers (Ambion) were run on the same gel to determine product size. The gels were exposed to x-ray film (Fuji; Fisher Scientific) for 3 days using intensifying screens. The amount of Hp58 mRNA was measured from the optical density (NIH image software) for each sample, and values were divided by the optical density of the corresponding G3PDH standard in the same reaction. Adjusted mean values were calculated from triplicate samples on the same autoradiograph. Mean differences between steady-state mRNA levels were considered to be significant if the Mann-Whitney LL-test for nonparametric data gave a P value of <0.05.

Cell Culture

The cell lines (HRP/LRP) used in this study were isolated from the labyrinth region of the rat placenta at midgestation. The cells were grown in RPMI medium containing 10% fetal bovine serum and supplements as described. Confluent cells were collected in guanidine thiocyanate, and total RNA was extracted with acid phenol: chloroform.

Localization of H/358 in Placental Cell Lines

The placental cell lines were grown on gelatin-coated coverslips. Hp58 immunoreactivity was assessed using standard methods with some modifications. Briefly, cells on coverslips were fixed with 2% paraformaldehyde for 30 min at 4°C, washed, and nonspecific binding sites were blocked with Dulbecco-PBS containing powdered milk, gelatin, and Tween-20. Primary antibody (120 ^g/ml) and preimmune serum (122 ^g/ml) were applied to cov-erslips for 90 min at 22°C. The coverslips were washed in Dulbecco PBS and blocked with 5% goat serum for 30 min. Goat antirabbit-Oregon green secondary antibody (Molecular Probes, Eugene, OR) was diluted 1:100 and applied to coverslips for 60 min at 4°C. Random fields of cells were photographed using identical conditions for cells treated with preimmune serum and Hp58 antibody. Cells were photographed using an Olympus microscope equipped with an appropriate fluorescent filter and Tmax film.

top