Allele specific probes

Samples of 200 μl of anticoagulant-containing blood were collected according to the instructions of the Genomic DNA Extraction Kit. The specific procedures can be found in the literature [24–26 (link)]. The centrifuge tube was collected and quantified using a nucleic acid quantifier, which was eventually stored at <80°C. The miRNA-608 rs4919510 G>C polymorphism was genotyped with the TaqMan reagent. The allele-specific probes were purchased from Applied Biosystems. The PCR reaction was performed in 384-well plates that were run on an ABI-Q6 Sequence Detection System machine [27 ]. Moreover, to ensure the quality and accuracy of the genotyping results, we randomly selected 10% of the samples for a repeat analysis, and the results were 100% concordant.

Samples of 200 µl of anticoagulant-containing blood were collected according to the instructions of the Genomic DNA Extraction Kit. The specific procedures can be found in the literature [21–23 (link)]. The centrifuge tube was collected and quantified using a nucleic acid quantifier, which was eventually stored at −80°C. The miR-218 rs11134527 A>G polymorphism was genotyped with TaqMan reagent. Allele-specific probes were purchased from Applied Biosystems. The PCR reaction was performed in 384-well plates that were run on an ABI-Q6 Sequence Detection System machine [24 (link),25 (link)]. Moreover, to ensure the quality and accuracy of the genotyping results, we randomly selected 10% of the samples for repeat analysis, and the results were 100% concordant.

We had extracted Genomic DNA from 200 μL of blood collected from each participant using a TIANamp Blood DNA Kit (Tiangen, Beijing city, China) and we followed the specific procedures in the literature (35 (link)–37 (link)). The extracted DNA is placed in a −80°C refrigerator until use. The allele-specific probes were purchased from Applied Biosystems. TaqMan real-time polymerase chain reaction of the samples is performed in 384-well plate with an ABI Q6 instrument (Thermo Fisher Scientific, Waltham, MA, United States) to genotype rs7404339 polymorphism in CDH5 (38 (link), 39 (link)). For quality control, each 384-well plate contained eight samples without DNA but with the same amount of distilled water. Moreover, to ensure the quality and accuracy of the genotyping results, we randomly selected 10% of the samples for a repeat analysis, and the results were 100% concordant.