Study population

Our study population consisted of neonates of pregnant women who had been admitted to the hospital with a diagnosis of threatened preterm birth who had received at least 1 dose of antenatal corticosteroids. The women, admitted to 1 of 2 urban hospitals, were recruited to a study and gave consent for obtaining samples for DNA from their newborn infants. Women who consented were at least 23 weeks’ gestation and ≥18 years old.

Sample collection and processing

Blood samples. Umbilical cord blood samples were obtained immediately after delivery in K ₂ EDTA vacutainers by trained cord blood collectors. Samples were mixed by inversion 8-10 times after being drawn and then stored at −80 °C until DNA isolation.

Saliva samples. If umbilical cord blood was not acquired at the time of delivery because of an offhour delivery when the study personnel were not available, a trained member of the research team obtained buccal swabs from the neonate. For buccal cell collection, we used Oragene saliva kit collection with cotton swabs. Oragene kits are shown to reduce microbial contamination and provide immediate stabilization of samples, allowing it to be stored at room temperature for years without processing or DNA degradation. Oragene “Saliva collection with cotton swabs or buccal brushes” protocol was followed. Collection protocols call for collector to grip swab without contaminating cotton tip and gently place the cotton tips inside of the infant’s mouth, collecting as much saliva as possible by rubbing the cheeks and moving the tip into the spaces between the check and gums and under the tongue. Once saturated in saliva, tips were cut off of swabs into the base of the Oragene collection vial. This process was repeated until the 5 cotton tips that come with the collection kit were inside of the collection vial. Immediately on collection, the Oragene vial cap, which contains the Oragene · DNA solution was placed onto the base and tightened securely. The vial was shaken vigorously and inverted to ensure that the cotton tips were well mixed with the Oragene · DNA preservation fluid. Kits were then stored at room temperature. Before purification, the Oragene saliva sample kits were briefly mixed by gentle inversion and incubated for a minimum of 2 hours in an air incubator set to 50°C. To remove saliva from buccal swabs, manufacturer protocols for “DNA Recovery from Saliva Sponges” were followed. First, free liquid was removed and transferred into a 15-mL conical centrifuge tube. A barrel of a 5-mL disposable plastic syringe (with the plunger removed) was placed into the same tube. Sterile, disposable forceps were used to transfer the sponges from the kit collection base into the barrel of the syringe. The syringe barrel containing the sponges in the tube was centrifuged at 200× g for 10 minutes at 20°C. The barrel containing the dry sponges was discarded and the tube containing the recovered liquid was capped, labeled, and stored at room temperature until purification.

DNA isolation

DNA was extracted from umbilical cord blood samples using the QIAamp DNA mini, midi, or maxi kits (Qiagen Inc, Valencia, CA). The specific kit used was chosen based on sample volume collected and period of collection. As with the buccal swab samples, we originally extracted only a portion of the sample collected but later began extracting the full sample to attain maximal DNA yield. Kits are designed to extract the following sample volumes: mini (0.2 mL), midi (1-2 mL), and maxi (3-5 mL). Initially, all samples were extracted using the minikit. Subsequent samples were extracted using the kit whose volume capacity best fit the total sample volume collected. Manufacturer spin protocol instructions were followed for all kits with one modification. Our equipment was not capable of reaching 4500× g for large tube sizes. For steps where the manufacturer protocol called for centrifugation at 4500× g , we used 3100× g and compensated this difference by using increased spin times. When manufacturer’s protocols listed steps for highly concentrated DNA, those steps were followed. Isolated DNA was transferred into cyrovials and all samples were stored at −80°C until quantification.

DNA was purified from saliva swab samples using the Oragene Kit (DNA Genotek) manufacturer recommendations. Two procedures were used throughout the course of the study. Initially, 500 μL of sample was transferred to a 1.5-mL Eppendorf tube and purified. However, we found DNA yield insufficient using only a portion of the collected sample prompting us to extract the entire sample volume collected from subsequent participant samples. Oragene saliva kits are designed to collect 2 mL saliva, which are then added together with 2 mL Oragene · DNA present in the cap of each kit resulting in 4 mL total sample to be extracted. However, using the “saliva collection with cotton swabs or buccal brushes” and “DNA recovery from saliva sponges” protocols, only minimal volumes of saliva were recovered (≤200 μL). Regardless of sample volume that used DNA isolation was performed according to manufacturer’s instructions. Samples were frozen at −80°C until quantification.

DNA quantification

Concentration of double-stranded DNA in our samples was determined using a Quant-iT double-stranded DNA (dsDNA) broad range or high-sensitivity assay kit and Qubit Flouromter (Invitrogen, Carlsbad, CA). This system uses a fluorescent nucleic acid stain to accurately and specifically measure dsDNA at highly sensitive levels.

DNA quality assessment by gel electrophoresis

The DNA quality was assessed by analyzing the samples for evidence of degradation using gel electrophoresis. Samples of both buccal and umbilical cord blood genomic DNA (180 ng each sample) were run on an agarose gel (0.9% agarose) and stained with ethidium bromide. The size of the DNA was determined by comparison with a DNA ladder (1 kb; Life Technologies). The largest band in the ladder is approximately 10 kb. High-quality DNA is expected to be mostly >10 kb.

Statistical analysis

All results were analyzed with SPSS, version 17.0 (SPSS, Inc, Chicago, IL). Nonparametric tests were used to compare mean total yield and concentration of DNA collected from Oragene buccal swab collection samples and umbilical cord blood samples because the samples were not normally distributed. We used the Mann-Whitney U test. We also compared the DNA concentration between extraction protocols for partial sample extraction and complete sample extraction. For the purposes of comparison umbilical cord blood and buccal swab samples were each separated into 2 groups based on whether the entire blood or buccal cell sample collected or a portion of the entire sample collected was isolated in this study. In addition, a linear regression analysis was performed to compare the possible effect of gestational age at cord blood collection and day of life at buccal swab collection on DNA yield measures.