Postmortem RNA and protein stability in perinatal human lungs.
The availability of fetal and neonatal lung tissue is an invaluable resource to elucidate the molecular regulation of human lung development. In this study, we have investigated the mRNA and protein stability of perinatal lung tissues treated with RNA (Ambion Inc., Austin, TX) or snap frozen in liquid nitrogen (LN ). Lung samples were obtained from 25 consecutive perinatal autopsies of live-born and stillborn infants (median gestational age, 23 weeks) with various clinical presentations. Treatment of lung tissue with RNA yielded more total RNA and protein than LN freezing. The integrity of RNA, assessed by spectrophotometry and gel electrophoresis, was equivalent between both tissue preservation methods, and both methods produced RNA suitable for reverse transcriptase-polymerase chain reaction analysis of representative genes (beta-actin and surfactant protein-B [SP-B]). Similarly, the protein integrity of RNA -treated tissues was equivalent to that of LN -frozen tissues, as judged by Western blot analysis of SP-B/actin protein expression. Although the total yield was similar in live-born, nonmacerated stillborn and macerated stillborn infants, only RNA and protein from live-born or nonmacerated stillborn infants was suitable for subsequent molecular analyses. Within the 41-hour range studied, the duration of the postmortem interval did not affect the yield or integrity of RNA and protein with either tissue preservation method. In summary, high-quality RNA and protein, suitable for routine molecular analyses, can be obtained from postmortem lung tissue from live-born and nonmacerated stillborn infants, even with prolonged postmortem intervals. RNA is equivalent, if not superior, to LN for preservation of postmortem RNA and protein in developing human lungs.
De Paepe
Monique E
ME
Department of Pathology, Women and Infants' Hospital, Providence, RI 02905, USA. mdepaepe@wihri.org
Mao
Quanfu
Q
Huang
Carol
C
Zhu
Danping
D
Jackson
Cynthia L
CL
Hansen
Katrine
K
D001329:Autolysis; D015925:Cryopreservation; D004355:Drug Stability; D005313:Fetal Death; D005865:Gestational Age; D006801:Humans; D007231:Infant, Newborn; D008168:Lung / Q000196:embryology / Q000254:growth & development / Q000378:metabolism*; D019188:Organ Preservation Solutions; D011506:Proteins / Q000378:metabolism*; D037701:Pulmonary Surfactant-Associated Protein B / Q000032:analysis / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors
D019188:Organ Preservation Solutions; D011506:Proteins; D037701:Pulmonary Surfactant-Associated Protein B; D012313:RNA