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Real-time quantitative PCR analysis of Nannochloropsis gaditana

ABSTRACT: Nannochloropsis gaditana cells were cultivated under continuous light as a control condition versus 3 different flashing light conditions (FL5, FL50 and FL500). This study is complementary to Lima et al. (2020) where physiology and photosynthetic capacity were assesed. Here we analyzed the mRNA expression level of key genes related to photoshynthesis, lipid and starch synthesis, and nitrogen assimilation. Total cell RNA was extracted from cultures of N. gaditana at a concentration of 1x106 cells/mL using the E.Z.N.A® total DNA/RNA isolation kit (Omega Bio-tek, Inc., USA). Reverse transcription reactions were performed using SuperScript™ IV VILO™ Master Mix with the enzyme ezDNase (Invitrogen™, ThermoFisher scientific, USA) according to the manufacturer’s protocol. The qPCR reactions were performed by mixing 5L of FastStart Universal SYBR Green Master mix (Rox) (Roche Molecular Systems, Inc., USA) with 1 L of primer mix (consisting of forward and the reverse primers) at a concentration of 300 nM and 4 L of the cDNA. The conditions of thermocycling were: 95 C for 600 s (preincubation), followed by 40 cycles of denaturation at 95 C for 10 s, annealing/extension at 60 C for 30 s. qPCR were performed in the LightCycler 96 Roche Life Science instrument.

ORGANISM(S): Nannochloropsis gaditana

PROVIDER: GSE210188 | GEO | 2022/08/02

REPOSITORIES: GEO

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Project description:Human Peripheral blood mononuclear cells were purified from older volunteers' blood and were cultured for 24 hrs in the presence or abscence of angiotensin type 1 receptor autoantibody (AT1RaAb 7ug/ml). In brief: PBMCs were isolated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation (86). PBMCs from older individuals cultured in six-well plates (0.5 x 106 cells/ml) were divided into two groups: control group and treatment group. The treatment group was incubated with purified AT1R-aAb (Eagle bioscience, Noshua, New Hampshire) for 24 hours. Supernatant was collected and cells harvested after a total culture period of 24 hours. RNA extraction, RT2 profiler assay and pathway analysis: Control and AT1R-aAb treated monocytes and lymphocytes from participants were prepared for RNA isolation. Total cellular RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany). cDNA was synthesized at 37°C for 60 minutes and the reaction was stopped by heating the reaction mix to 94 °C. PCR cycling parameters are 10 minutes at 95°C and 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. All primers for the polymerase chain reaction (PCR) were purchased from Operon Technologies, Inc. (Alameda, CA). We performed quantitative PCR with an Mx-3000P Real Time PCR System Instrument and SYBR Green florescent reagents (Stratagene, La Jolla, CA). QPCR to determine AT1R expression was performed using the TaqMan gene expression assay mix (Applied Biosystems) after reverse transcription of RNA with the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA). To avoid DNA contamination in RT-PCR assays, samples were extensively processed using DNase 1 digestion. Gene expression profiling for inflammation and autoimmunity target genes was performed using RT2 profiler PCR arrays (SuperArray, Frederick, MD) following the manufacturer's instructions. In brief, 1 μg RNA was reverse transcribed with the RT2 profiler PCR array first strand synthesis assay (SuperArray, Frederick, MD) followed by real-time PCR with RT2 real-time PCR master mix Sybr green (SuperArray, Frederick, MD). PCR data from the treated and untreated cells were analyzed by the QIAGEN SuperArray group and the results reported as differential gene expression and statistical significance, fold change and p-value respectively.

Project description:Diabetes Mellitus (DM) is a chronic, severe disease rapidly increasing in incidence and prevalence and is associated with numerous complications. Patients with DM are at high risk of developing diabetic foot ulcers (DFU) that often lead to lower limb amputations, long term disability, and a shortened lifespan. Despite this, the effects of DM on human foot skin biology are largely unknown. Thus, the focus of this study was to determine whether DM changes foot skin biology predisposing it for healing impairment and development of DFU. Foot skin samples were collected from 20 patients receiving corrective foot surgery and, using a combination of multiple molecular and cellular approaches we performed comparative analyses of non-ulcerated non-neuropathic diabetic foot skin (DFS) and healthy non-diabetic foot skin (NFS). MicroRNA (miR) profiling of laser captured epidermis and primary dermal fibroblasts from both DFS and NFS samples identified 5 miRs de-regulated in the epidermis of DFS though none reached statistical significance. MiR-31-5p and miR-31-3p were most profoundly induced. Although none were significantly regulated in diabetic fibroblasts, miR-29c-3p showed a trend of up-regulation, which was confirmed by qPCR in a prospective set of 20 skin samples. Gene expression profiling of full thickness biopsies identified 36 de-regulated genes in DFS (>2 fold-change, unadjusted p-value ≤ 0.05). Of this group, three out of seven tested genes were confirmed by qPCR: SERPINB3 was up-regulated whereas OR2A4 and LGR5 were down-regulated in DFS. However no morphological differences in histology, collagen deposition, and number of blood vessels or lymphocytes were found. No difference in proliferative capacity was observed by quantification of Ki67 positive cells in epidermis. These findings suggest DM causes only subtle changes to foot skin. Since morphology, mRNA and miR levels were not affected in a major way, additional factors, such as neuropathy, vascular complications, or duration of DM, may further compromise tissue’s healing ability leading to development of DFUs. samples were fixed in formalin and paraffin embedded. Between 16 and 20 8-10µm sections were cut from the formalin-fixed paraffin embedded tissue blocks of non-diabetic and diabetic foot skin, placed on Arcturus PEN-membrane glass slides (Life Technologies, Carlsbad, CA, USA) and dried at 37°C for 1-2 hours. LCM was carried out on a Arcturus Veritas laser capture microdissection instrument and the epidermis was collected on CapSure® Macro LCM Caps (Life Technologies). The caps were transferred to a tube containing 60µl of deparaffinization buffer (QIAGEN Inc., Valencia, CA, USA) and total RNA, including the microRNA fraction, was extracted using the FFPE miRNeasy kit (QIAGEN Inc.) according to the manufacturer’s instructions. Total RNA concentration of the samples was quantified using NanoDrop 2000 (NanoDrop products, Wilmington, DE) and the RNA quality of these samples was determined by RT-qPCR of SNORD48 and miR-21 using the commercially available platforms miRCURY LNA™ (Exiqon, Woburn, MA, USA) or Quanta qScript™ microRNA Quantification System (Quanta BioSciences, Inc., Gaithersburg, MD, USA). The miR profiles for the epidermis of 3 NFS and 3 DFS, were generated using the miR Ready-to-Use PCR panels V3 (Exiqon) following the manufacturer’s specifications. The Ct values were normalized to the stably expressed reference gene SNORD49 using the Exiqon GenEX software and the expression levels in the NFS and DFS were compared.

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Real-time quantitative PCR analysis of Nannochloropsis gaditana

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