Morning fasting venous blood samples from donors were collected for RNA extraction. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by Ficoll gradient separation. In order to achieve lysis and inactivate endogenous, RNAses Cells were mixed with mirVana RNA lysis buffer (Ambion, Austin, Texas, USA). Then the lysates were frozen at -80°C until RNA purification.
Extracted molecule
total RNA
Extraction protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions.
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions。
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=5μm, PMT 100%, 10%, 16bit.
Description
Gene expression in whole blood from patient B6 with low viral load LVL_B6
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US). With a total of 37 samples, the raw data from all of the 37 samples was handled within the R statistical computing framework version 3.0.2 (http://www.R-project.org) to obtain the processed data. As for miRNA raw data, R package AgiMicroRna providing useful functionality for the following processing, quality assessment for Agilent microRNA array data was employed used. In AgiMicroRna, the processed microRNA signal can be obtained using total Gene Signal (TGS) computed by the AFE and RMA algorithm. There were altogether 499 probes with corresponding miRNA in subsequent analysis after the normalization. As well as for mRNA, R package Agi4x44PreProcess was used for background correction, normalization and filtering probes according to different quality flags during the microarray standardization in our study. Ultimately, total 28155 probes mapping 14507 genes denoted by ENTREZ ID were obtained between arrays.
Identification of HIV1-specific cascaded microRNA-mRNA regulatory relationships by parallel mRNA and microRNA expression profiling with AIDS patients after antiviral treatment
