RNA purification was performed using the Arcturus PicoPure RNA Isolation Kit (#KIT0202, KIT0204), including digestion of genomic DNA using a RNase free DNase set (QIAGEN, #79254). RNA concentration was measured with a Nanodrop Spectrophotometer (Peqlab; ND-1000, RRID:SCR_016517 ); 20 ng purified RNA was used for reverse transcriptase-mediated cDNA synthesis, using the Thermo Fisher Scientific First Strand cDNA Synthesis Kit (#K1612). cDNA was diluted 1:5 in RNase free H2O, and 2 µl cDNA was used for qRT-PCR using a Roche Diagnostics LightCycler 96 with the following primers against total BDNF (forward: 5'-AAATTACCTGGATGCCGCAAAC-3'; reverse: 5'-CGCTGTGACCCACTCGCTAA-3') and mouse GAPDH (forward: 5'-GCAAATTCAACGGCACA-3'; reverse: 5'-CACCAGTAGACTCCACGAC-3'). Results were exported to Microsoft Excel and analyzed with GraphPad Prism 6.0 for statistical analysis. BDNF values were normalized to GAPDH, and BDNF mRNA levels were calculated in percent of mean BDNF mRNA levels in P21 sedentary mice.
Nd 1000
Manufactured by Avantor
Sourced in Germany
The ND-1000 is a spectrophotometer designed for accurate and reliable UV-Vis absorbance measurements. It offers a wide wavelength range, high resolution, and precise quantification capabilities. The ND-1000 is a versatile instrument suitable for a variety of applications in laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Picopure rna isolation kit, by Qiagen (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (1 mentions)
Sequence detection software 2, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Bamhi hf, by New England Biolabs (1 mentions)
6 protocols using nd 1000
1
Quantifying BDNF mRNA Expression in Mice
2
RNA Extraction and qRT-PCR Analysis
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RNA was isolated using a NucleoSpin RNA II kit (Macherey-Nagel) and quantified using the NanoDrop method (ND1000, Peq lab). Primers for quantitative real-time PCR are given in Table S5 . mRNA expression data were normalized to the housekeeping Gapdh gene and calculated relative to the respective unstimulated controls.
3
Genotyping of D2 and D3 Receptor SNPs
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DNA from buccal swabs or buffy coat was isolated using the QIAamp DNA Blood Mini Kit (Cat No. 51106, Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. Concentration and quality of the DNA were assessed with an UV/vis spectrophotometer (ND-1000, Peqlab GmbH, Erlangen, Germany). For SNP genotyping of rs6280 (D3 receptor), 20 ng of DNA was analysed using allelic discrimination assays (TaqMan SNP Genotyping Assays, Applied Biosystems by Thermo Fisher Scientific Inc., Waltham, MA, USA). SNP genotyping for rs1800497 (ANKK1) was performed with 20 ng of DNA in triplicates using allelic discrimination assays (TaqMan SNP Genotyping Assays, Applied Biosystems by Invitrogen). Genotyping polymerase chain reaction (PCR) was performed on a 7900HT Fast Real-Time PCR system (Applied Biosystems) and the data analysed with Sequence Detection Software (SDS) 2.3 (Applied Biosystems). Groups were defined for the D2 receptor according to ANKK1 protein [A1− (wild type): GG; A1+: AG, AA] and for the D3 receptor [B1− (wild type): TT; B1+: TC, CC].
4
Quantification of AQP5 mRNA in Septic Patients
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The RNA samples collected from whole blood were considered eligible for analysis when their concentration was greater than 50 ng/µl, according to nanodrop measurement (ND-1000, PEQLAB Biotechnologie GmbH, Erlangen, Germany). The RNA samples from 98 septic patients were available in acceptable quality. First-strand cDNA was synthesized from 0.5 µg of total RNA using a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The qPCR reaction was performed using GoTaq® qPCR Master Mix (Promega, Mannheim, Germany), as described previously5 (link). A cDNA dilution series for AQP5 confirmed a PCR efficiency >95%, which was comparable to the efficiency of ß-actin (data not shown). Relative AQP5 mRNA expression was measured by two-step qPCR and expressed as 2-∆CT.
5
Cloning sh-miR204-5p Plasmid
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For the second cloning step, both the new plasmid containing the sh-miR204-5p cassette and puromycin (pLKO-sh-miR204-5p-puro), and a donator plasmid (pLKO-U6-DsRed) were double-digested with BamHI-HF (R3136S; New England BioLabs) for 30 min at 37 °C and SfiI (R0123S; New England BioLabs) for 30 min at 50 °C with CutSmart Buffer according to the manufacturer’s recommendations. The fragments were separated within a TAE gel containing 1.5% agarose and the 1.3 kb DsRed from the donator pLKO-U6-DsRed well as the 7.6 kb BamHI/SfiI band (marker: 1kb plus Ladder) from the backbone were excised and DNA was extracted using the PeqGOLD Gel Extractions Kit (732-2777; PeqLab, VWR Chemicals) following the manufacturer’s protocol. DNA concentration was measured photometrically (ND 1000, PeqLab) and ligation was performed at RT for 1 h with T4-DNA Ligase and 5× Ligation Buffer.
6
Optimizing Antibody-Coated AuNP Stability
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For the visualization of the tested biomarker, AuNP-labeled detection antibodies were part of the sandwich assay in the LFA. In order to find the lowest antibody concentration necessary to completely cover the gold nanoparticles and create stable particles, a flocculation test was performed.
During the flocculation test, possible aggregations of gold nanoparticles due to the addition of NaCl as the electrolyte solution were formed. Aggregation generally occurs, if gold nanoparticles are not fully covered by antibodies, and can be photometrically visualized as a color change. 46 Within the flocculation test, 20 µL of AuNPs and different volumes of the detection antibody were adjusted to a final volume of 23 µL by the addition of sodium borate buffer. Solutions were incubated in the dark for 20 min at 115 rpm at room temperature. The absorption spectrum was measured using NanoDrop (Peqlab ND-1000) from 300 to 745 nm. 47, 48 (link)
During the flocculation test, possible aggregations of gold nanoparticles due to the addition of NaCl as the electrolyte solution were formed. Aggregation generally occurs, if gold nanoparticles are not fully covered by antibodies, and can be photometrically visualized as a color change. 46 Within the flocculation test, 20 µL of AuNPs and different volumes of the detection antibody were adjusted to a final volume of 23 µL by the addition of sodium borate buffer. Solutions were incubated in the dark for 20 min at 115 rpm at room temperature. The absorption spectrum was measured using NanoDrop (Peqlab ND-1000) from 300 to 745 nm. 47, 48 (link)
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