Miniaturized Workflow for Transcriptomic Profiling of Urinary Extracellular RNA during Pregnancy.

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Urine contains extracellular RNA (exRNA) within extracellular vesicles (EVs) and other complexes.
• There is a need for non-invasive biofluid analysis methods, particularly in female cohorts, for reproductive and pregnancy disorder biomarkers.
• Urinary exRNA profiling can reflect pregnancy-associated changes in maternal and placental tissues.

Purpose of the Study:

• To identify optimal methods for transcriptomic profiling of urinary extracellular RNA (exRNA).
• To test different exRNA isolation and scalable library preparation techniques.
• To enable the detection of biomarkers for pregnancy-associated conditions.

Main Methods:

• RNA extraction from pooled and individual urine samples (non-pregnant, pregnant females, males) using varying input volumes (0.6 mL, 1 mL, 4 mL).
• Isolation methods focused on vesicular (EV-associated) or total (EV-associated and non-EV-associated) exRNA.
• Small RNA libraries (n=208) prepared with NEBNext Small RNA kit; long RNA libraries (n=97) with SMART-Seq v4 or SMARTer Stranded Total RNA-Seq Kit v2 Pico.

Main Results:

• Principal component analysis indicated exRNA isolation method as the primary variance source for small RNA libraries, and library preparation method for long RNA libraries.
• Long RNA libraries from miRCURY-extracted exRNA showed SMART-Seq v4 yielded significantly more uniquely mapped reads than Pico v2 (p<0.05).
• A scalable, reproducible pipeline for urine exRNA small and long RNA-Seq was established.

Conclusions:

• The developed pipeline enables reproducible transcriptomic profiling of urinary exRNA.
• This methodology can identify differentially expressed urinary exRNAs during pregnancy.
• The pipeline will facilitate transcriptomic profiling for disorders of pregnancy, such as preeclampsia.