Gene expression was determined by real-time quantitative PCR, using TaqMan detection systems (Applied Biosciences). For in vitro experiments, RNA was extracted using the High Pure RNA Isolation kit (Roche) and RNA quality was assessed by Nanodrop spectrometry (Thermo Fisher). Total RNA from isolated organs (spleen and vagina) was extracted by use of the Qiagen RNase Plus Mini Kit (Qiagen) according to the manufacturer's guidelines and RNA quality assessed by Nanodrop spectrometry (Thermo Fisher). mRNA levels for murine Ifnb1 (Mm00439552_s1), Cxcl10 (Mm00445235_m1), Tmem173 (Mm01158117_m1), Ifit2 (Mm00492606_m1), Nfe2l2 (Nrf2) (Mm00477784_m1), and β-Actin (Mm02619580_g1) were analyzed using premade TaqMan assays (Thermo Fisher) and the RNA-to-Ct-1-Step kit according to the manufacturer's recommendations (Applied Biosciences).
Nanodrop spectrometry
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nanodrop spectrometry is a laboratory equipment used to measure the concentration and purity of nucleic acid and protein samples. It utilizes a specialized optical technology to analyze small sample volumes, typically in the range of 1-2 microliters, without the need for cuvettes or dilution.
Automatically generated - may contain errors
Lab products found in correlation
High pure rna isolation kit, by Roche (4 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Sybr green pcr master mix, by Bio-Rad (3 mentions)
Tripure isolation reagent, by Roche (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Aceq qpcr sybr green master mix, by Vazyme (2 mentions)
Charcoal agar plates, by Thermo Fisher Scientific (1 mentions)
Mmessagemmachine t7, by Thermo Fisher Scientific (1 mentions)
Sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
Mycycler thermal cycler, by Bio-Rad (1 mentions)
Trizol, by Takara Bio (1 mentions)
Truseq small rna sample preparation kit, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Magattract hmw dna kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
17 protocols using nanodrop spectrometry
1
Quantitative PCR Analysis of Murine Genes
2
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from fresh left hemisphere samples using the TriPure Isolation Reagent (Roche, South San Francisco, CA, USA) according to the manufacturer's instructions, and the concentration of RNA was measured by Nanodrop spectrometry (Thermo Fisher Scientific). RNA quality was determined with the OD 260/280 ratio, which was between 1.8 and 2.0. Up to 0.5μg of RNA was used to synthesize the first strand of cDNA using PrimeScript™ RT Reagent Kit (TaKaRa, Kusatsu, Shiga, Japan). Reverse transcription products were amplified with the 7900HT Fast Real-Time PCR System in a 10 μl final reaction volume using SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) under the following conditions: 2 min at 95°C and 30 s at 60°C, followed by a total of 40 cycles of 2 temperature cycles (15 s at 95°C and 30 s at 60°C) [66 (link)]. The forward and reverse primer sequences are shown in Table 1 . The fluorescence threshold value (Ct value) was calculated using the SDS Enterprise Database software. The Ct values of the interested genes were first normalized with β-actin of the same sample, and then the gene expression level in sham and melatonin-treated groups were calculated and expressed as fold change versus sham group (setting sham as 1).
3
DNA Extraction and Qualification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was isolated after proteinase K digestion using a DNA extraction kit (Cinnagene, DN8115C), according to the manufacturer's instructions
Then isolated DNA was purified and qualified via nanodrop spectrometry (Thermo Fisher Scientific, USA).
Then isolated DNA was purified and qualified via nanodrop spectrometry (Thermo Fisher Scientific, USA).
4
Bacterial DNA Extraction from B. pertussis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Strains were stored at −80 °C in PBS/20 % glycerol at the University of Bath. They were grown on charcoal agar plates (Oxoid) for 72 h at 37 °C. All cells were harvested from each plate and resuspended in 3 ml PBS. The optical density of each cell suspension was measured at 600 nm, and volumes of suspension equating to an OD of 1.0 (~2×109B. pertussis cells) in 180 µl were pelleted in a microcentrifuge for 2 min at 12 000 g . gDNA was extracted from each pellet using a GenElute bacterial genomic DNA kit (Sigma Aldrich) according to the manufacturer’s instructions, including the optional RNAase A step and a two-step elution into 200 µl elution buffer (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0). QuBit fluorometry (dsDNA HS kit; Invitrogen) was used to measure gDNA concentration, and Nanodrop spectrometry (ThermoFisher Scientific) was used to assess gDNA purity.
5
DENV and ZIKV Reporter Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids for DENV or ZIKV vectors which encode GFP (pFK-DVs-G2A for DENVGFP), Renilla luciferase (pFK-DVs-R2A, serotype 2, strain 16681 for DENVR2A; pFL-ZIKV-R2A strain FSS13025 for ZIKVR2A) reporter genomes, wild-type (WT) DENV (pFK-DVs; 16681), subgenomic (sg) replication-deficient NS5 mutant (sg-DVs-R2A-GND), and WT (sg-DVs-R2A-WT) subgenomic replicon systems were all obtained from Ralf Bartenschlager and Pei-Yong Shi. Plasmids were linearized with XbaI (DENV) or ClaI (ZIKV) and purified, and 1 μg was in vitro transcribed using the mMESSAGE mMACHINE T7 or SP6 transcription kit (Invitrogen). The resulting RNA quality and concentration were assessed by migration on a 0.8% agarose gel and Nanodrop spectrometry (Thermo Scientific), respectively.
6
Quantitative RT-PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the samples using the TriPure Isolation Reagent (Roche, South San Francisco, CA, USA) according to the instructions provided by the manufacturer. NanoDrop spectrometry (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the concentration of total RNA; only samples with an optical density 260/280 ratio >1.8 were selected. RNA (0.5 μg) was used to synthesize the cDNA utilizing the PrimeScript™ RT Reagent Kit (TaKaRa, Kusatsu, Japan) and Bio-Rad MyCycler™ thermal cycler for the reverse transcription–polymerase chain reaction. With the use of the SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA), samples were amplified by the real-time polymerase chain reaction system. β-Actin was used for standardization. The forward and reverse primer sequences are shown in Table 1 . Subsequently, we calculated the fluorescence threshold value (cycle threshold value) via the SDS Enterprise Database software. The cycle threshold values of the correlative genes were normalized to those of β-actin in the same sample. Next, the levels of gene expression (fold change) in the vehicle- and liraglutide-treated groups were measured and compared with those reported in the sham group.
7
Quantitative Analysis of Zebrafish Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of zebrafish was extracted using Trizol (Takara), and quantitated by NanoDrop spectrometry (Thermo Scientific, Wilmington, DE, USA). cDNA was generated by MMLV reverse transcriptase (Invitrogen). Real-time PCR was performed using AceQ® qPCR SYBR® Green Master Mix (Vazyme) according to the manufacturer’s instructions and relative gene expression was quantified using the StepOnePlusTM Real-Time PCR System (Life Technologies). Gene primers are listed in Supplementary Table 2 .
8
Exosomal Small RNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from exosome isolate using miRNeasy Mini Kit (Qiagen, cat# 217004). Total RNA concentration was measured using Nanodrop spectrometry (Thermo Fisher Scientific).
Small RNA libraries were prepared using TruSeq Small RNA Sample Preparation Kit (Illumina, USA) with multiplexing adapters, following the kit user guide (Rev. E), and sequenced by MiSeq V3 flow cell using Illumina MiSeq reagent kit V3 for 150 cycles and 36 bp reads with single-end chemistry as previously described (Bourgery et al., 2021 (link)).
Small RNA libraries were prepared using TruSeq Small RNA Sample Preparation Kit (Illumina, USA) with multiplexing adapters, following the kit user guide (Rev. E), and sequenced by MiSeq V3 flow cell using Illumina MiSeq reagent kit V3 for 150 cycles and 36 bp reads with single-end chemistry as previously described (Bourgery et al., 2021 (link)).
9
Fibroblast Cell Culture and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All fibroblasts except the 241 cell* were seeded in 6-well culture plates at 1,4 105 cells/ml in a total volume of 2 ml DMEM 2% SVF. The next day, the medium was exchanged to DMEM 0,5% SVF for 48 h. Total RNA was extracted using the RNAeasy Mini kit (Qiagen) which included a RNAse-Free DNAse treatment step to eliminate DNA contamination and RNA quality was assessed by Nanodrop spectrometry (Thermo Fisher). Reverse transcription was performed using the SuperScript II Reverse Transcriptase (InVitrogen).
*The cells of donor 241 were lost.
*The cells of donor 241 were lost.
10
Zebrafish Total RNA Extraction and qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of zebrafish was extracted using Trizol (Life, 15596018), and quantified by NanoDrop spectrometry (Thermo Scientific, Wilmington, DE, USA). cDNA was generated using MMLV reverse transcriptase (Invitrogen, 28025013). Real-time PCR was performed using AceQ® qPCR SYBR® Green Master Mix (Vazyme, Q141-02/03) according to the manufacturer’s instructions, and relative gene expression was quantified using the Step One PlusTM Real-Time PCR System (Life Technologies).
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!