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Megalin expression in human term and preterm placental villous tissues: effect of gestational age and sample processing and storage time.

Author(s): Akour AA, Gerk P, Kennedy MJ

Publication: J Pharmacol Toxicol Methods, 2015, Vol. 71, Page 147-54

PubMed ID: 25304941 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects gestational age on the stability of megalin expression in placental villous tissue. Placental tissue from healthy pregnant adults (18 to 45 years) delivering by Cesarean section at term (10 cases ≥ 36 weeks) and preterm (5 cases < 36 weeks) were stored snap-frozen in liquid nitrogen. Placental villous tissue fragments were prepared after cutting and draining the umbilical cord by removing the basal and chorionic plates, processed by several cycles of mincing and washing in antibiotic-supplemented Dulbecco's phosphate buffered saline (DPBS), and frozen in 300–400 mg aliquots for up to 4 months. RNA was isolated using the Trizol method and megalin mRNA expression was measured by TaqMan real-time RT-PCR analysis and levels were normalized using 18S as the reference gene.

   Summary of Findings:

   Megalin mRNA levels in placental villous specimens were strongly correlated with gestational age (r2=0.74, P=0.05), with mean levels 4.95 and 5.26-fold higher in early pre-term (<32 weeks) than in moderate pre-term (32-35 weeks) or term specimens, respectively (P= 0.05 for both). Megalin mRNA levels were approximately 6-fold higher in early pre-term specimens compared to those from moderate pre-term specimens.

   Biospecimens

   • Tissue - Placenta

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	term pregnancy (≥ 36 weeks) preterm pregnancy (< 36 weeks) early preterm pregnancy (<32 weeks) moderate pre-term (32-35 weeks)

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2. Study Purpose

   This study investigated the effects of storing processed placental villous tissues at 4 °C for 1, 2, 4, 6 or 18 h before or after frozen storage at −80 °C on mRNA levels of Megalin. Placental villous tissue was prepared from 6 healthy pregnant adults delivering by Cesarean section at term by draining the umbilical cord by removing the basal and chorionic plates, processed by several cycles of mincing and washing in antibiotic-supplemented Dulbecco's phosphate buffered saline (DPBS). Specimens were stored at 4˚before or after snap-freezing in liquid nitrogen and frozen storage at -80˚C for up to 4 months. RNA was isolated using the Trizol method and megalin mRNA expression was measured by TaqMan real-time RT-PCR analysis and levels were normalized using 18S as the reference gene.

   Summary of Findings:

   Megalin mRNA expression in placental villous tissue snap-frozen immediately after processing was approximately 1.5-fold higher than in specimens stored at 4°C after processing for 1, 2, 4, 6 and 18 h prior to freezing (P=0.05); however, there were no significant differences in mRNA levels among the specimens stored prior to freezing for any of the time points. In contrast, megalin mRNA expression declined with post-thaw storage such that levels in snap-frozen specimens were 3.4, 6.25, 6.25, and 33.3-fold higher in specimens analyzed immediately after thawing than in specimens thawed at 4 °C for 1, 2, 4, 6 and 18 h prior to analysis, respectively (P=0.05).

   Biospecimens

   • Tissue - Placenta

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Thaw duration	1 h 2 h 4 h 6 h 18 h
   Storage	Storage duration	1 h 2 h 4 h 6 h 18 h

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3. Study Purpose

   This study investigated the effects of storing placenta tissue at 4°C before or after processing and storage at -80˚C on megalin mRNA levels. Placental villous tissue was prepared from 6 healthy pregnant adults delivering by Cesarean section at term by draining the umbilical cord by removing the basal and chorionic plates, processed by several cycles of mincing and washing in antibiotic-supplemented DPBS. Specimens were stored at 4˚before or after processing and then frozen in liquid nitrogen and storage at -80˚C for up to 4 months. RNA was isolated using the Trizol method and megalin mRNA expression was measured by RT-PCR analysis and levels were normalized using 18S as the reference gene.

   Summary of Findings:

   Megalin mRNA expression was significantly higher in specimens processed prior to storage at 4 °C than in specimens that were stored unprocessed for all time points examined (P=0.05). There was no significant difference in megalin levels between specimens that were processed and snap-frozen immediately and those that were processed and stored at 4°C storage for 1, 2, 4, 6, 10, or 18 h before freezing; however, there was a significant decrease when processed specimens were stored for 24 and 48 h before freezing (P=0.05). There was a significant correlation between storage time at 4°C prior to processing and megalin expression (P= 0.05) with a 40% decrease occurring within the first hour of storage.

   Biospecimens

   • Tissue - Placenta

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	1 h 2 h 4 h 6 h 10 h 24 h 48 h
   Storage	Storage conditions	pre-processing post-processing

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