Illumina HiSeq 2000 sequencing; Whole-blood MicroRNA Sequence Profiling for Adolescents with Postural Tachycardia Syndrome
Whole-blood MicroRNA Sequence Profiling for Adolescents with Postural Tachycardia Syndrome
From September to December 2018, five children with POTS and five healthy children were recruited from the outpatient pediatric department of the Children's hospital of Zhejiang University School of Medicine, China. All five POTS children underwent a thorough medical history evaluation, physical examination, and laboratory assessment, including electrocardiography (ECG), electroencephalography, blood glucose and blood biochemistry analyses, and cranial computed tomography or magnetic resonance imaging to exclude cardiac, neurologic, metabolic, and psychogenic causes. Five healthy children, matched as far as possible in terms of age, gender, height, weight, and residence location, were recruited as control subjects. The healthy controls were screened by medical history assessment, physical examination, and laboratory assessment, including ECG, Holter ECG, and standing tests. These 10 children were used as sequencing groups. Another 40 POTS patients and 20 healthy children from Shenmu County Hospital and Shaanxi Provincal People's Hospital were used as a validation subjects. All children were informed about the purposes of the research and agreed to participate. Written informed consent was obtained from the parents or guardians of all the study subjects. The study was approved by the ethics committees of the Children's Hospital of Zhejiang University School of Medicine, Shenmu County Hospital, and Shaanxi Provincial People's Hospital. Blood sample collection for whole-blood miRNA sequencing POTS patients and healthy children were required to fast before blood sampling. Blood samples (5 mL) were drawn at 8:00 am from supine subjects into PAXgene Blood RNA Tubes (BD). The tubes were inverted 8–10 times gently immediately after blood collection. Each tube contained 2.5 mL of whole blood and 7 mL of additives. The blood samples were allowed to stand at room temperature for 4 h and then stored at -80 ℃.   Total RNA extraction We extracted RNA from the PAXgene Blood RNA tubes and performed quality control. All the quality control results were analyzed centrally at the end of 2018. All the samples were thawed at room temperature, agitated, and observed to determine the presence of any blood clots in the tubes. The PAXgene Blood RNA kit and a QIA cube were used to extract RNA from the samples automatically. RNA was eluted with elution buffer and stored at -80 °C.  A total of 2 μg of RNA/sample was used as input material for the miRNA library. The miRNA sequencing libraries were generated using a TruSeq Small RNA Sample Preparation Kit (Illumina, Inc., San Diego, CA, USA), and index codes were added to attribute sequences to each sample. Briefly, an RNA 3′ adaptor was added to the total RNA and the mixture was incubated at 70 °C for 2 min to break the RNA secondary structure and then immediately placed on ice. Then, the RNA 3′ adaptor was specifically ligated to the 3′ end of the miRNA with T4 RNA Ligase 2 and T4 Rnl2 truncated at 28 °C for 1 h. After the 3′ ligation reaction, the ligated fragment was further ligated to an RNA 5′ adapter using T4RNA ligase at 28 °C for 1 h. The first strand cDNA was then synthesized using SuperScript IV (SS IV) according to the manufacturer's instructions. Polymerase chain reaction (PCR) amplification was performed using 5× Phusion HF buffer, RNA PCR Primer (RP1), RNA PCR Primer Index (RPLX) primer, dNTP, and Phusion DNA polymerase. The PCR products were purified on a 6% polyacrylamide gel. RNA fragments corresponding to 140–160 bp (the length of small noncoding RNA with the 3′ and 5′ adaptors) were recovered and dissolved in 8 μL of elution buffer. Finally, the library concentration was measured using a Qubit Spectrophotometer and the library quality was assessed using an Agilent 2100 Bioanalyzer system with a High Sensitivity DNA Kit (Agilent Technologies). The clustering of the index-coded samples was performed on an Illumina cBot cluster station using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2000 platform and 50 bp single-end reads were generated for transcriptome analysis. The quality control of the samples, experiments, and data analysis were completed at Genesky Biotechnologies Inc. (Shanghai, People's Republic of China).