Urine exosome purification and rna isolation midi kit

Composite samples (pools) were established prior to the isolation of exosomes and the amplification of miRNAs, mixing equivalent amounts of individual samples. A total of 5 pools containing 34 individual samples and 5 pools containing 27 individual samples were established from the healthy controls and GDM groups, respectively. Each pool was matched by age and body mass index (BMI).
Exosomes were purified from 10 ml of pooled urine samples using the Urine Exosome Purification and RNA Isolation Midi kit (Norgen Biotek Corp, Product # 58700) following the manufacturer's instructions. The Urine Exosome Purification kit isolates exosomes based on the isoelectric point of the proteins present in the microvesicles that are filtered through silicon carbide columns, which makes the method sensitive and specific (19 ). Purified exosomes were stored at -80°C for subsequent analysis.

Total RNA was isolated from 400 µl of purified exosomes using the Urine Exosome Purification and RNA Isolation Midi kit (Norgen Biotek Corp.; cat. no. 58700) following the manufacturer's instructions. The RNA concentration was assessed using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies Inc.). The cDNAs for the mature miRNAs (U6, miR-16-5p, miR-222-3p, miR-516b-5p, miR-517-5p and miR-518a-3p) were synthesized from 150 ng of total RNA using the Taq Man^® microRNA test, (Applied Biosystems); TaqMan probes were used for each of the miRNAs to avoid unspecific amplification. The 5'-3' sequences of the primers are presented in Table SIII. The CFX96 touch thermal cycler was used (Bio-Rad Laboratories, Inc.).
All RT-qPCR reactions were performed in duplicate; Cq values were averaged and the 2^−ΔΔCq method (20 (link)) was used to obtain the relative expression, where ^ΔΔCq was calculated by subtracting the ^ΔCq value from the mean of the control group (healthy pregnant women) with the ^ΔCq value of GMD women. miRNA levels were normalized with the use of the average of U6 snRNA Cq value as the housekeeping gene. The experimental conditions for reverse transcription and PCR are detailed in Tables SIV and SV, respectively.