gender: Male age: 56 years disease state: non-cardiac chest pain (control) tissue: plasma
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from plasma or tissues using miRNeasy Mini Kit (Qiagen, Valencia, CA). 250 µl of EDTA-plasma were mixed with 700 µl Qiazol, incubated for 5 min at room temperature and subsequently mixed with 140 µl chloroform. The organic and aqueous phase was separated by centrifugation at 12,000 g for 15 min. The upper aqueous phase was collected and the RNA was precipitated by adding 100% ethanol. The mixture was applied to a miRNeasy Mini spin column. After washed several times, the RNA was eluted in 25 µl RNase-free water.
Label
FAM
Label protocol
Five ul total RNA per sample was reverse-transcribed using Megaplex primers, sets A and B (Applied Biosystems). cDNA was pre-amplified for twelve cycles with Mexaplex Pre-Amp primers, sets A and B (Applied Biosystems), then diluted to 100 ul. Manufacturer's protocol was followed throughout. Total RNA was reverse-transcribed, pre-amplified with miRNA-specific primers, and loaded into TLDA cards (2 per sample). Quantitative real-time PCR was performed for 768 features, 754 of which represent primate miRNAs For each of two cards per sample (A and B), nine ul of pre-amplified, diluted sample was mixed with TaqMan reaction mix (Applied Biosystems) and loaded into miRNA TLDA cards (Applied Biosystems) by centrifugation. Cards were sealed, and qRT-PCR was performed with a real-time thermocycler (ABI-7900, Applied Biosystems) per manufacturer's recommendations.
Hybridization protocol
n/a
Scan protocol
n/a
Description
SAMPLE 2
Data processing
SDS software (Applied Biosystems) was used to collect data. Threshold intensities were set, visually and individually confirmed, and standardized with RQ Manager (Applied Biosystems). Data were processed using DataAssist (Applied Biosystems). For the normalization it uses Mamm U6 as housekeeping gene. Raw miRNA expression CT values were normalized to the endogenous housekeeping gene MammU6; 2^-deltaCt, where deltaCt = (Ct_Target - Ct_Mamm U6)
