A RNeasy kit was used to extract total RNA from hMOs on days 0, 7, 15, and 25 (Qiagen, Valencia, CA, USA). Total RNA was reverse transcribed (Quantitech, Qiagen) for qRT-PCR analysis. ABI Prism (Applied Biosystems, Waltham, MA, USA) was used for the qRT-PCR analysis. GAPDH was used as the reference gene.
Quantitech
Manufactured by Qiagen
Sourced in United Kingdom
The QuantiTech is a real-time PCR system designed for quantitative analysis of nucleic acid samples. It is capable of performing sensitive and accurate quantification of target molecules in a wide range of applications, including gene expression analysis, pathogen detection, and DNA/RNA quantification.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Quantitative real time polymerase chain reaction, by Thermo Fisher Scientific (1 mentions)
Icycler iq, by Bio-Rad (1 mentions)
Ambion pure link kit, by Qiagen (1 mentions)
T7 polymerase, by Roche (1 mentions)
Topo ta vector, by Thermo Fisher Scientific (1 mentions)
Dig rna labeling mix, by Roche (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
5 protocols using quantitech
1
Quantitative RNA Expression Analysis
2
Gene Expression Analysis in Rat Mesenteric Arteries
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Quantitative real-time Polymerase Chain Reaction (Applied Biosystems) was used to analyze mRNA expression in rat mesenteric arteries; data are expressed as target gene/18 s housekeeping gene. Transcript expression in rat VSMCs was normalised to GAPDH, using Qiagen QuantiTech primer assays (Qiagen, UK). Relative gene expression was calculated by 2ΔΔCt method, and the results were reported as arbitrary units relative to the control conditions. Primers were designed using Primer3 software with sequences as shown in Table 1 .Table 1
Primers targeted to rat genes for qRT-PCR analysis.
| Rat gene | Forward primer | Reverse primer |
|---|---|---|
| Nox1 | TCCCTTTGCTTCCTTCTTGA | CCAGCCAGTGAGGAAGAGTC |
| NoxA1 | TTACTGTGCCCCTGAAGGTC | CTCGGGCTTTGTAGCTGAAC |
| NoxO1 | TCCAGACGTTTGCCTTCTCT | CGTGTCAACAATGGAGCATC |
| Nox2 | ACCCTTTCACCCTGACCTCT | TCCCAGCTCCCACTAACATC |
| Nox4 | CCAGAATGAGGATCCCAGAA | AGCAGCAGCAGCATGTAGAA |
| P22phox | TTGTTGCAGGAGTGCTCATC | CAGGGACAGCAGTAAGTGGA |
| P47phox | AGCTCCCAGGTGGTATGATG | ATCTTTGGCCGTCAGGTATG |
| MMP2 | AGCTCCCGGAAAAGATTGAT | TCCAGTTAAAGGCAGCGTCT |
| MMP9 | CACTGTAACTGGGGGCAACT | CACTTCTTGTCAGCGTCGAA |
| MCP-1 | CAGTTAATGCCCCACTCACC | TTCCTTATTGGGGTCAGCAC |
| RANTES | ATATGGCTCGGACACCACTC | CCACTTCTTCTCTGGGTTGG |
| 18S | AAGTCCCTGCCGTTTGTACACA | GATCCGAGGGCCTCACTAAAC |
3
Lipid Analysis in Perfused Mice
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All mice were culled by exsanguination under terminal anaesthetic (isoflurane > 4% in 95% O 2 5% CO 2 ); depth of anaesthesia was monitored by respiration rate and withdrawal reflexes. Biochemical analyses of plasma lipids were performed on heparinised blood plasma using enzymatic assays. Tissue for histological analysis was collected from mice perfused with phosphate buffer saline (PBS) followed by 4% paraformaldehyde, tissue for biochemical analysis was collected from mice perfused with PBS only and was snap frozen in liquid nitrogen and stored at -80 °C until analysis.
Total RNA was extracted using the Ambion Pure Link kit, RNA was reverse transcribed using the QuantiTech reverse transcription kit (Qiagen), quantitative real-time RT-PCR was performed with an iCycler IQ real-time detection system (BioRad Laboratories). Gene expression data were normalized to an appropriate house keeper using the delta CT method.
Total RNA was extracted using the Ambion Pure Link kit, RNA was reverse transcribed using the QuantiTech reverse transcription kit (Qiagen), quantitative real-time RT-PCR was performed with an iCycler IQ real-time detection system (BioRad Laboratories). Gene expression data were normalized to an appropriate house keeper using the delta CT method.
4
Cloning and RNA probes generation
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cDNA was generated using QuantiTech (Qiagen) with RNA extracted (Qiazol) from S1 to S14 P. tepidariorum embryos and from S7 to S17 for P. opilio. Gene fragments were amplified by PCR and cloned into the TOPO-TA vector (ThermoFisher Scientific). Primer sequences are provided in supplementary table 1 , Supplementary Material online. RNA probes were transcribed with T3 (11031163001—Roche) or T7 polymerase (10881775001—Roche), with DIG RNA labeling mix (11277073910—Roche), from PCR fragments generated from TOPO-TA clones following standard protocols.
5
Temporal Gene Expression Analysis
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Total RNA at days 0, 30, 45, and 60 of each of the line were reverse transcribed (Quantitech, Qiagen) and amplified material was detected using commercially available Taqman gene-expression assays (Applied Biosystems) with the data normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT). Each data point represents nine technical replicates from three independent experiments.
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