Developmental expression of phospholipase D2 mRNA in rat brain

Abstract

Phospholipase D (PLD) is a ubiquitous enzyme involved in many important cellular functions. Rat brain PLD isoforms have recently been cloned and characterized, but the expression of this gene has not been well studied. We thus examined the developmental expression of PLD2 in postnatal brains by Northern blotting employing a non-radioactive RNA probe. Two PLD2 mRNA transcripts of 3.9 and 10.8 kb were detected in different brain regions and various tissues of the rat. In postnatal brains, the level of PLD2 mRNA transcripts are low 1 day after birth, increase progressively during development, and reach the maximum level in the adult brain. We thus conclude that rat brain PLD2 mRNA is developmentally regulated.

Down-regulation of Phospholipase D2 mRNA in Neonatal Rat Brainstem and Cerebellum after Hypoxia-Ischemia

“…Detailed procedures regarding RNA extraction, electrophoresis, membrane blotting, riboprobe preparation, and Northern-blot hybridization have been reported [20]. In brief, brain tissues after removing from sacrificed rat pups were quickly transferred to TRI Reagent (Molecular Research Center), homogenized, and total RNA samples were isolated.…”

“…Non-radioactive riboprobes of PLD2 and b-actin were transcribed respectively from PLD2 clone and b-actin cDNA (Ambion) in the presence of Biotin-14-CTP (life Technologies) using Maxiscript kit as previously described [20]. Northern hybridization was carried out by prehybridizing the RNA blot in a buffer containing 5· SSC, 5· Denhardt solution, 50 mM sodium phosphate, pH 6.8, 5 mM EDTA, 1% SDS, and 0.1 M NaCl for at least 1 h at 68°C, and the buffer was removed.…”

“…A pre-warmed hybridization buffer containing 5 ng/ml of PLD2 riboprobe was added and incubated at 68°C overnight for 16 h. The hybridized blot was washed with a 1· SSPE-1% SDS buffer three times and then with a solution of 0.2· SSC-0.1% SDS twice at 68°C, each washing for 15 min. After removing unbound riboprobe, the blot was treated with blocking solution for 1 h, then with streptavidin-alkaline phosphatase conjugate (1:10,000) for 1 h, and the hybridization signal was detected using a Biodetect kit (Ambion) as described [20]. Experimental data were evaluated by one-way analysis of variance and Student's 2-tailed t-test using SigmaStat to determine the significance of data.…”

“…It is because the mechanisms leading to cell death in these areas after cerebral HI injury has so far received little attention and requires more insight. Thus, it is taking advantage of the PLD2 riboprobe previously prepared [20] to test the above hypothesis by looking into whether PLD2 transcription is altered after HI insult.…”

PLD2 (phospholipase D2)

“…Specially, PLD2 mRNA has been detected in many different brain regions (Peng and Rhodes, 2000). During rat brain development, PLD2 mRNA levels are elevated and peak in the adult brain.…”