To isolate RNA from the human abdominal SAT, a piece of the tissue was homogenized in 700 µL of TRIsure reagent (Bioline, USA) using the bead-based tissue homogenizer PowerLyzer24 (MO BIO Laboratories, USA). The genomic DNA (gDNA) in the lysate was removed using a gDNA eliminator spin column and the RNA was purified using RNeasy plus micro kit (Qiagen, no: 74034) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA with a high-capacity cDNA reverse transcription kit (Applied Biosystems, no: 4368814), and gene expression was analyzed using a FastStart Universal SYBR green master mix (Sigma-Aldrich, no: 04913914001) kit. The gene expression assay was performed in Bio-Rad C1000 thermal cycler according to the standardized protocol of the quantitative PCR master mix supplier. The quantification cycle (Cq) values of the technical triplicates were collected using Bio-Rad CFX Manager software (v3.1). The data were imported to qBase plus software (Biogazelle, https://www.qbaseplus.com ), and the Cq average of each sample were normalized to the housekeeping genes 36B4 and YWHAZ. The mRNA expression level was calculated and presented as fold a change (control=1). The human primer sequences are listed in online supplementary material .
Qbaseplus software
Manufactured by CellCarta
Sourced in Belgium
QbasePLUS software is a data analysis platform designed for scientific and research applications. It provides tools for processing and visualizing data from various types of laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (9 mentions)
Trizol, by Thermo Fisher Scientific (8 mentions)
Lightcycler 480, by Roche (6 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (5 mentions)
Gotaq polymerase, by Promega (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Rneasy plus micro kit, by Qiagen (3 mentions)
Dnase enzyme, by New England Biolabs (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Steponeplus machine, by Thermo Fisher Scientific (2 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (2 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
85 protocols using qbaseplus software
1
RNA Isolation and Quantification from Adipose Tissue
2
Analyzing Gene Expression Profiles by qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The results obtained from reference genes study were analyzed by the qBasePLUS software (Biogazelle). Experiments to evaluate gene expression profile by qPCR were performed from three independent biological replicates and the results were represented as average ± Standard Deviation of gene relative expression. The unpaired two-tailed Student’s t-test (GraphPad, https://www.graphpad.com/quickcalcs/ttest1 ) was applied to verify statistical significance of the observed variation. Significant variations were defined as *P < 0.05.
3
Quantitative Gene Expression Analysis by RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression analysis was done by quantitative two-step reverse transcription PCR. Reverse transcription was performed using total RNA and a High Capacity cDNA Reverse Transcription kit (Life Technologies), using random hexamers. Quantitative PCR (qPCR) was done using the Fast SYBR Green Master Mix (Life Technologies) with specific primer pairs (Supplementary Table SII ). For each target mRNA analysed, 2.5 μl of Fast SYBR Green Master Mix, 0.5 μM of each primer pair and 2 μl of cDNA in deionised water (5 ng/μl) were mixed in 384-well plates in duplicates using Matrix Equalizer Electronic Multichannel Pipetters (Thermo Fisher Scientific, Loughborough, UK). qPCR was carried out on an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA) using the following settings: initial activation of 20′′ at 95 °C, 40 cycles; denaturation for 1′ at 95 °C; annealing/extension for 20′′ at 60 °C; final melting curve was carried out for 15′′ at 95 °C and then for 15′′ at 60 °C. Quantification of target messages was performed using qbasePLUS software (Biogazelle, Ghent, Belgium). HPRT and GAPDH were the reference genes used for normalisation.
4
Relative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Relative gene expression for each investigated gene was calculated using qBasePlus-Software (Version 1.3, Biogazelle) or the method of Pfaffl.36 Primer efficiency was determined for each primer by a cleaned up PCR-product dilution series (QIAquick PCR Purification Kit, Qiagen). HPRT1 was chosen as a reference gene for normalization of relative gene expression of each gene. It was selected as the most stable gene of all tested genes by ‘NormFinder’-algorithm.37 (link) Stability value was calculated as 0.200±0.094.
5
Quantitative PCR Analysis of Ephrin Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were sacrificed by cervical dislocation and cortical tissue was collected as described above (western blot paragraph). Total RNA was extracted using Tripure (Roche) and isopropanol (Sigma-Aldrich) purification. cDNA was synthesized using the SuperscriptTM III First-Strand Synthesis Mastermix kit (Thermofisher Scientific) and quantitative PCR reactions were performed using 5 μl cDNA and predesigned qPCR assays (IDT). Thermal cycling was done on a StepOne-Plus Real-Time PCR system (Applied Biosystems, Foster City, USA) using a standard amplification protocol with Taqman assays (IDT). Data were processed and analyzed in qBase Plus software (Biogazelle, Gent, Belgium). The following 20× assays were used: Epha4 (Mm.PT.58.13545379), Efna1 (Mm.PT.58.29850712), Efna2 (Mm.PT.58.11885886), Efna3 (Mm.PT.58.10380528), Efna4 (Mm.PT.58.9770779), Efna5 (Mm.PT.58.28681125), Efnb1 (Mm.PT.58.28819484), Efnb2 (Mm.PT.58.29108694), Efnb3 (Mm.PT.58.41654515), Polr2a (Mm.PT.58.13811327), Ywhaz (Mm.PT.39a.22214831) and Gapdh (Mm.PT.39a.1).
6
qPCR Analysis of Osteoblast Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For qPCR analyses RNA was isolated from total tibia lysates using the Trizol Reagent (Invitrogen), reverse transcribed using the high capacity cDNA transcription kit (life technologies). The qPCR was carried out using the TaKaRa Syber Premix Ex Taq-kit on an Eppendorf Mastercycler epRealplex machine. qPCR analyses have been established following the MIQE guidelines36 (link) applying 3 reference genes for callibrating the relative expression level. Primer-sequences are listed in supplementary Table S1 .Data was analysed using the qBase Plus software (Biogazelle). Results are expressed as a ratio of the mean expression level in OPN deficient cells compared to the WT controls. Statistical analysis was done using the unpaired two-tailed student t-test.
7
RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted with the RNeasy Plus Micro Kit (Qiagen, 74034). cDNA was synthesized using the High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Quantitative PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, 4367659) following the manufacturer's protocol. The primers used in qPCR are listed in Appendix Table S2 . GAPDH, 36b4, and TBP were used as internal expression controls. The results (CNRQ; calibrated normalized relative quantity) were analyzed with qbase Plus software (Biogazelle, Gent, Belgium).
8
Quantitative Real-Time PCR Assay for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative real-time PCR reactions were performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., CA, USA) using the KAPA SYBR® FAST qPCR Master Mix Universal Kit (KAPA Biosystems) reagent. Three biological replicates were run in technical triplicates. The reaction mixture (10 μL) was composed of 5 μL of qPCR Master Mix (2x), 0.25 μL of each primer (10 μΜ), 2 μL of diluted cDNA template (12-fold), and 2.5 μL of Nuclease-free water. The qPCR cycling conditions were initial denaturation at 95 °C for 3 min followed by 40 cycles of 95 °C for 3 s, 60 °C for 30 s, and 72 °C for 15 s. Gapdh and rpl13 (geNorm kit, PrimerDesign) were used as internal controls. Normalization and relative quantification of gene expression were performed with the qBase Plus software (Biogazelle). Primers for the genes of interest were designed based on Danio rerio nucleotide sequences found in NCBI, their Tm was 60 °C and their sequences are listed in Table 3 .
9
Quantitative Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression analysis was performed according to previously described method (Demuyser et al., 2017 (link)). The cells were grown to mid-exponential phase in SC medium supplemented with the carbon source of interest. Cells were washed with ice-cold Milli-Q water, frozen in liquid nitrogen and kept at −80°C. The cell pellet was dissolved in TRIzol (Thermo Fisher), and cells were lysed by fast prepping with glass beads. Afterward, RNA was isolated using chloroform, isopropanol, and 70% ethanol. The RNA was treated with DNase enzyme (New England Biolabs), to remove present DNA fractions, and converted into cDNA by using the iScript cDNA synthesis kit (Bio-Rad). To perform the quantitative PCR, a Go-Taq polymerase (Promega) and a StepOnePlus machine (Thermo Fisher) were used. The results were analyzed by making use of the qBasePlus software (Biogazelle).
10
Quantitative RT-PCR of Neural Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neurospheres were collected before plating on PLL (undifferentiated NSCs) or after differentiation for 1 or 4 days. RNA was extracted using the RNeasy Micro Kit (Qiagen) and quantified with a Nanodrop-1000 Spectrophotometer (NanoDrop Technologies, Inc). qRT-PCR was performed as described in ref. 36 (link). Complementary DNA was analyzed by qRT-PCR (5 ng RNA equivalent/reaction) using 0.2 µM target-specific primers and QuantiFast SYBR Green (Qiagen). Primers were validated by qRT-PCR analysis on serial dilutions of a positive control complementary DNA to assess their specificity and efficiency. Forty qRT-PCR cycles were performed on 7900HT instrument (Applied Biosystems) equipped with the Sequence Detection Systems (SDS) 2.3 software. Data were normalized using the expression of the housekeeping genes Actin and HPRT1 as in ref. 36 (link). Ct values were converted into fold-expression values relative to the control using qBasePLUS software (Biogazelle).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!