Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling†. (7th November 2016) - Pisarska, Margareta D.
Abstract: Background: Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. Results: Leftover CVS samples were flash‐frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2–5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA‐sequencing (RNA‐seq). RNA‐seq libraries generated using 200 versus 800‐ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite‐conversion and genome‐wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. Conclusions: CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd. Abstract : What's Already Known About This Topic? Chorionic villus sampling (CVS) tissue is used for diagnostic testing of genetic disorders. Because of sample size, CVS specimens are limited to single platform testing. What Does this Study Adds? Our refined techniques outlined for proper storage and processing of direct CVS specimens give us the ability to conduct genetic, epigenetic and gene expression studies on a single sample that can be used for the development of diagnostics and potentially treatments using a systems biology approach for conditions that manifest early in gestation.