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| Status |
Public on Apr 01, 2024 |
| Title |
Peripheral blood mononuclear cells (PBMCs) 1b was obtained from the subjects before the third dose of CoronaVac |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral blood samples were obtained from the subjects before the third dose of CoronaVac.
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: PBMC
|
| Treatment protocol |
RNA was prepared using the Versagene Blood RNA Purification kit (Gentra Systems, Minneapolis MN) following the manufacturer's recommendations. The protocol includes differential lysis of red and white blood cells, and an on-column DNase digestion. Globin message was further reduced using GLOBINclear (Ambion Inc., Austin, TX) to specifically remove both a- and b- globin. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Growth protocol |
Percoll density gradient followed by plastic adherence was used for PBMC separation. Nine milliliters of heparinized peripheral blood from healthy volunteers was mixed with 9 ml phosphate-buffered saline (PBS). Aliquots of the mixture (6 ml) were overlaid onto 6 ml Ficoll-Paque density gradient (Cedarlane, Burlington, NC, USA) in 15 ml tubes, which were then centrifuged at 2000 rpm for 20 min. Peripheral blood mononuclear cells (PBMC) at the interface were collected and washed twice with PBS.
|
| Extracted molecule |
total RNA |
| Extraction 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, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
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| Hybridization protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
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| Scan protocol |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| Description |
Peripheral blood samples 1b was obtained from the subjects before the third dose of CoronaVac,total RNA was extracted from PBMC
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
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| Submission date |
Nov 30, 2023 |
| Last update date |
Apr 01, 2024 |
| Contact name |
guanguan qiu |
| E-mail(s) |
qiuguanguan88@126.com
|
| Phone |
15005853326
|
| Organization name |
shaoxing second hospital
|
| Street address |
Yanan Road123
|
| City |
shaoxing |
| State/province |
zhejiang |
| ZIP/Postal code |
312000 |
| Country |
China |
| |
|
| Platform ID |
GPL24741 |
| Series (1) |
| GSE249050 |
Altered expression of PBMC miRNA profile following the third dose of inactivated COVID-19 vaccine |
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