Venous Blood Case Studies

Venous blood samples (~5 ml) were collected from normal healthy subjects (n-10) and patients presenting with NSTEMI (n=50) on admission. Based on the duration between the onset of chest pain symptoms and hospital admission, samples were divided into three groups such as hospital admission within 1 hour ( ≤1 hr), or after 1 hour but within 4 hours ( ≥1 hr -≤4 hrs) and after 4 hours but within 8 hours (≥4 hrs- ≤8 hrs) after the onset of chest pain. Half of the blood sample was allowed to stand overnight at 4˚C before being centrifuged at 1500 × g for 10 min to obtain serum (Schaeffer et al, 2000). The resultant serum was aliquoted into RNase/DNase-free tubes and stored at -80°Cuntil RNA extraction.
For the separation of plasma, about 2.5 ml blood was added into potassium-ethylene-diaminetetraacetic acid (K2-EDTA)coated tubes. Plasma was separated by centrifugation at 1,000xg for 10 minutes at 4˚C to remove blood cells, followed by centrifugation of the supernatant at 14,000 x g for 10 minutes at 4˚C to remove additional cellular nuceic acids attached to cell debris. The plasma was transferred into RNase/DNase-free tubes and stored at -80 ˚C until RNAextraction. …show more content…
Reverse transcription of miRNA was performed with the miScript reverse transcription kit (Applied Biosystem). miR-499 was quantitated by using TaqMan miRNA quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay according to the protocol of the manufacturer (Applied