Stability of placental RNA using dried maternal blood spots.
Having demonstrated successful recovery and detection of placental transcripts from dried blood spots (DBS), various preanalytical conditions were examined to determine optimal handling of samples. The role of several factors was explored, including temperature (4 degrees C versus 25 degrees C), processing time (24 h to 8 weeks), and addition of preservatives (RNA later and formalin) that may interfere with stability and detection of placental transcripts in DBS. mRNA transcripts encoding human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal control) and beta-human chorionic gonadotrophin (beta HCG; placental) were analysed by real-time-polymerase chain reaction using DBS from 23 pregnant women. GAPDH and beta HCG transcripts were detected in all samples 24 h after collection. Although treatment of blood with RNA later did not affect RNA recovery, formalin treatment negatively affected RNA recovery from DBS. Temperature did not have a significant effect on levels of either transcript. Storage time caused a significant decrease in GAPDH after 4 weeks (P = 0.014) and beta HCG after 1 week (P = 0.007). Decrease of beta HCG levels after 1 week followed by steady detectable levels for up to 4 weeks suggests two populations of circulating placental transcript exist, a population susceptible to degradation in blood versus a more stable form. Therefore, defining proper parameters for collection and storage of DBS further reinforces reliable analysis of target sequences for clinical testing.
Jorgez
Carolina J
CJ
Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, USA.
Bischoff
Farideh Z
FZ
D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics; D005260:Female; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D010920:Placenta / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D020871:RNA Stability; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification
D018997:Chorionic Gonadotropin, beta Subunit, Human; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases