An average of 2.5 ml of venous blood was collected into sodium heparin glass vacutainers from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of hospital discharge at the University of Rochester and the University at Buffalo and shipped to a central laboratory in Rochester. Freshly purified PBMCs were isolated by Ficoll gradient (Amersham Pharmacia Biotech # 17-1440-03) centrifugation, from the whole blood diluted 1:2 with 1x dPBS, and counted according to previously established protocols (39 (link)). In subjects with at least 8 million cells, 5 million cells were stained with antibodies to individual lymphocyte markers, and sorted on a FACSAriaII sorter at the Flow Cytometry Core facility of the University of Rochester as previously reported (16 (link)). CD3+CD8+, CD3+CD4+, CD3-CD56+ (NK), or CD3-CD19+ (B) cells were collected separately. Non-marker positive and dead cells were discarded. Sorted cells were spun into pellets, which were further lysed and frozen. The steps from collection to lysis of each sample were completed within a 24-h period in order to preserve RNA quality and integrity. Frozen lysates were thawed and RNA was extracted using Agilent Absolute RNA Microprep kit (catalog #400805), with an on-column DNase digestion, as per manufacturer recommended protocol.
Absolute rna microprep kit
Manufactured by Agilent Technologies
The Absolute RNA Microprep kit is a laboratory product designed for the isolation and purification of total RNA from small samples. It is a tool for researchers to extract and concentrate RNA from various biological sources.
Automatically generated - may contain errors
Lab products found in correlation
Dnaase, by Merck Group (2 mentions)
Dispase 2, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Scgb1a1 ccsp, by LifeSpan BioSciences (2 mentions)
Foxj1 hfh4, by Novus Biologicals (2 mentions)
Pneumacult ali medium, by STEMCELL (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Collagenase type a, by Roche (2 mentions)
Elastase, by Worthington (2 mentions)
Ficoll gradient, by Cytiva (1 mentions)
Cdna synthesis kit, by Meridian Bioscience (1 mentions)
Dna engine opticon 2, by Bio-Rad (1 mentions)
Cfx manager software v3, by Bio-Rad (1 mentions)
Prism software v4, by GraphPad (1 mentions)
Cfx96 384 instruments, by Bio-Rad (1 mentions)
Gotaq qpcr master mix kit, by Promega (1 mentions)
4 protocols using absolute rna microprep kit
1
Isolation and Characterization of Immune Cells from Preterm Infants
2
Isolation and Characterization of Airway Epithelial Cells
3
Isolation and Characterization of Airway Epithelial Cells
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Collagenase type A (Roche, Basel, Switzerland); Dispase II (Gibco/ThermoFisher, Waltham, MA); Elastase (Worthington-Biochem, Lakewood, NJ); DNAase (Sigma-Aldrich, St. Louis, MO); Absolute RNA Microprep kit (Agilent, #400805, Stratagene, La Jolla, CA); bronchial epithelial basal medium (BEBM, Lonza, Mapleton, IL); small airway epithelial cell growth medium (SAGM; Lonza); Dulbecco’s modified Eagle medium (DMEM; GIBCO, Rockville, MD); PneumaCult-ALI medium (Stemcell Technologies, Vancouver, Canada); iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA); SYBR Green (Applied Biosystems, Foster City, CA. SCGB1A1/CCSP (Secretoglobin family 1A, member 1) antibodies (LifeSpan BioScience, Seattle, WA); FOXJ1/HFH4 (Novus Biologicals, Littleton, CO).
4
Quantitative Real-Time PCR Analysis
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RNA was isolated from ARPE-19, HDF cells and podocytes using an Absolute RNA Microprep Kit (Agilent Technologies). 500 ng RNA was used to generate cDNA using a cDNA synthesis kit (Bioline). Real-time qPCR analysis was carried out using either DNA Engine Opticon 2 (MJ Research Inc.) or CFX96/384 instruments (Bio-Rad) and the GoTaq qPCR Mastermix Kit (Promega). Expression analysis was performed in triplicate using CFX Manager software v3.0 (Bio-Rad), with samples normalised to a combination of TATA box binding protein (TBP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression unless otherwise stated. Gene expression data (relative to control cell expression) from across replicate experiments was either entered directly into Prism software v4.03 (GraphPad Software Inc.) or (when stated in figure legends) first imported into the ‘Gene Study’ functionality of CFX Manager prior to transfer of expression, s.e.m., and n-values into Prism. Prism software was used for analysis via two-way ANOVA with Bonferroni post-tests. The oligonucleotide primers used for all qPCR reactions are shown in supplementary material Table S1 .
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