Time course analysis of RNA stability in human placenta.
BACKGROUND
Evaluation of RNA quality is essential for gene expression analysis, as the presence of degraded samples may influence the interpretation of expression levels. Particularly, qRT-PCR data can be affected by RNA integrity and stability. To explore systematically how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples was generated by two protocols handlings of fresh tissue over a progressive time course of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing solution (RNAlater) solution. Protocol B uses a dissection of placenta villosities before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.


RESULTS
A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues that underwent a dissection before RNA processing by comparison with those transferred directly into RNA later solution. That loss is moderate, with average RIN (RNA Integration Numbers) range values of 4.5-6.05, in comparison with values of 6.44-7.22 in samples directly transferred to RNAlater (protocol A). Among the house keeping genes tested, the B2M is the most stable.


CONCLUSIONS
This study shows that placental samples can be stored at + 4 degrees C up to 48 h before RNA extraction without altering RNA quality. Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater) is a prerequisite to obtain suitable RNA integrity and stability.
Fajardy
Isabelle
I
Centre de Biologie Pathologie, Pôle de Biochimie et Biologie Moléculaire, CHRU de Lille, Université Lille 2, France. i-fajardy@chru-lille.fr
Moitrot
Emmanuelle
E
Vambergue
Anne
A
Vandersippe-Millot
Maryse
M
Deruelle
Philippe
P
Rousseaux
Jean
J
D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D010920:Placenta / Q000737:chemistry*; D011247:Pregnancy; D020871:RNA Stability / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors