tissue: peripheral blood cell type: CD4+ T-lymphocytes disease state: exposed to HIV-1 stage of hiv infection: antiretroviral therapy naive
Extracted molecule
total RNA
Extraction protocol
Purified CD4+ T-lymphocytes were stored in RNAlater at -80°C or directly processed for miRNA extraction by the mirVana Isolation Kit (Ambion).
Label
FAM
Label protocol
A total of 100 ng of RNA from CD4+ T cells was reverse transcribed using the TaqMan® MicroRNA Reverse Transcription Kit in combination with the Megaplex™ RT Primers Pool A, which permits analysis of 377 human microRNAs of greatest interest to the research community (functionally defined and/or broadly/highly expressed) and an endogenous control. The thermal profile consisted of 40 cycles at 16°C for 2 minutes, 42°C for 1 minute and 50°C for 1 second followed by one step at 85°C for 5 minutes. The pre-amplification of the miRNA cDNA target (2,5 μl) was performed using TaqMan® PreAmp Master Mix Kit and Megaplex™ PreAmp Primers Pool A (Applied Biosystems). The thermal profile consisted of denaturation at 95°C for 10 minutes, one step at 55°C for 2 minutes followed by 2 minutes at 72°C, and 12 cycles at 95°C for 15 sec and 60°C for 4 minutes. According to manufacturer’s instructions, the preamplified cDNA product was mixed with the TaqMan® Universal PCR Master Mix, No AmpErase UNG, and loaded onto the TaqMan® MicroRNA Array A for PCR amplification. The following real-time PCR protocol was employed: 50°C for 2 minutes, 94.5°C for 10 minutes and 40 cycles at 97°C for 30 seconds and 59.7°C for 1 minute. All reactions (reverse transcription, preamplification and real-time PCR) were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems).
Hybridization protocol
n/a
Scan protocol
n/a
Description
Purified_CD4+
Data processing
Amplification signal was checked on each sample by SDS® software 2.3. Ct value was determined using auto-baseline and a threshold value of 0.2. Undetermined raw Ct values were set to 40. The small non-coding MammU6 RNA was used as housekeeping gene, while the RNA extracted from the pool of healthy donors was used as calibrator.The data were analyzed using Relative Quantification Manager 1.2 (Applied Biosystems) Software, based on the comparative Ct method (∆∆Ct). The software package automatically performs all deltadeltaCt-based fold-change calculations from the uploaded raw threshold cycle data.
The supplementary file contains the SDS 2.3 RQ Results.
