Stability of expression of related factors mRNA in cryopreserved human amniotic membrane

Objective: Human amniotic membrane (AM) even cryopreserved for a long time has curative effects in many ocular surface diseases, but its treating mechanism is seldom to know. The aim of this research was to evaluate expression ability of several growth factors mRNA and cytokines in fresh or cryopreserved AM. Methods: Eight human placentas were obtained immediately after an elective cesarean section. AM was separated and cryopreserved. Fluorescein diacetate (FDA)/Propidium iodide (PI) combined with 0.5% trypan blue were used to identify epithelial cells viability. Total RNA was isolated from fresh AM and cryopreserved AM for 1, 2, 3, 4, 6 months separately. Real-time RT-PCR was used to evaluate mRNA expression levels of TGF-β₁, EGF, IL1β and IL1RA and compared to the endogenous control 18s rRNA. Results were expressed as 2ΔΔCT. Results: All the epithelial cells were dead in AM cryopreserved for 6 months. But numerous new cells outgrew from AM cryopreserved for 6 months after cell culture. The total RNA isolated from the different phases of cryopreserved AM did not show obvious degradation when the total RNA was extracted immediately after thawing (P > 0.05), but obviously RNA degradation occurred 1/2 hours after thawing (P <0.01). All the target mRNA can be amplified in cryopreserved AM in different phases with the same level as the fresh ones (P > 0.05). Conclusion: Traditional AM cryopreservation method seems to be a favorable way to keep the integrality of mRNA of growth factors or cytokines, and a new concept about genomic HAM bank should be established.