All sampling for RNA extraction was performed in the exponential phase of the E. coli strains with and without deipr_0871 under non-stressed and stressed conditions (0.45 mM H2O2), and without and with a high concentration of glucose (30 g/L). Total RNA was extracted using the RiboEx reagent (GeneAll, Seoul, Republic of Korea) supplied with DNase, and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA quality and concentration were estimated by measuring the optical densities of the solutions at 260 and 280 nm using a GeneQuant Pro instrument (Amersham Pharmacia Biotech, Amersham, UK). RNA (1 µg) from each sample was used for cDNA synthesis with random hexamers using a PrimeScript first-strand cDNA synthesis kit (TaKaRa Bio Inc., Kusatsu, Japan). RT-qPCR amplification was performed using SYBR Premix Ex Taq (TaKaRa) and an Eco Real-Time PCR system (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. The housekeeping gene polA was used as an internal control. Primers used for RT-qPCR are listed in Table S1 . All reactions were repeated in duplicates, and the relative expression was calculated using the relative quantification method.
Genequant pro
Manufactured by Cytiva
Sourced in United Kingdom, United States
The GeneQuant pro is a compact, full-spectrum spectrophotometer designed for accurate and reliable nucleic acid and protein quantification. It provides fast, simple, and reproducible measurements with a wide range of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Revertra ace, by Toyobo (4 mentions)
Sepasol rna 1 super, by Nacalai Tesque (3 mentions)
Ptc 100 programmable thermal controller, by Bio-Rad (3 mentions)
Rq1 rnase free dnase, by Promega (3 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Eco real time pcr system, by Illumina (1 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Riboex reagent, by GeneAll (1 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscopy, by Olympus (1 mentions)
Emx spectrometer, by Bruker (1 mentions)
Mda detection kit, by Nanjing Jiancheng (1 mentions)
Sepasol, by Nacalai Tesque (1 mentions)
Applied biosystems 2720 thermocycler, by Thermo Fisher Scientific (1 mentions)
Illustra rnaspin mini isolation kit, by GE Healthcare (1 mentions)
Dnase 1, by Promega (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Polytron pt 1200, by Kinematica (1 mentions)
Hiseq 2000 sequencing system, by Illumina (1 mentions)
18 protocols using genequant pro
1
RNA Extraction and RT-qPCR Analysis of E. coli
2
Thecal Layer Isolation and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thecal layers of F1 follicles were isolated and washed in PBS. Briefly, the superficial tissues were removed, followed by cutting the stigma to release the yolk and granulosa layer. The obtained thecal layer was separated into two pieces by cutting vertically, i.e., from the basal (where the stalk was located) to the apical stigma region. Total RNA was extracted from half of each theca using Sepasol RNA I Super (Nacalai Tesque Inc., Kyoto, Japan) and a Polytron™ homogenizer (Polytron™ PT1200ci Kinematica AG, Luzern, Switzerland). The RNA samples were then dissolved in TE buffer (10 mM Tris, pH 8.0, with 1 mM EDTA). The concentration of RNA in each sample was measured using Gene Quant™ Pro (Amersham PharmaciaBiotech, Cambridge, UK). The RNA samples were mixed with RQ1 RNase-free DNase (Promega Co., Madison, WI, USA) and incubated in a PTC-100 programmable thermal controller (MJ Research, Inc., Waltham, MA, USA) at 37°C for 45 min. The RNA samples were then reverse-transcribed using ReverTra Ace® (Toyobo Co., Ltd, Osaka, Japan) at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min on the PTC-100 programmable thermal controller (MJ Research, Inc.). The reaction mixture (10 µL) contained 1 µg of purified RNA, 1 × RT buffer, 1 mM dNTP mixture, 20 U RNase inhibitor, 0.5 mM oligo(dT)20 primer, and 50 U ReverTra Ace.
3
Quantitative RNA Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA extraction was then carried out. Briefly, harvested cells in suspension were adjusted to a concentration of 1×106/l, and inoculated into cell culture flasks. The cells were then treated in accordance with the above groupings. After 24 h, the cells were collected again and total RNA was extracted. Subsequently, 5 µl of RNA was added to 2% agarose gel for electrophoresis for detection of its quality, and the RNA concentration and purity were detected using ultraviolet spectrophotometry using GeneQuant Pro (Amersham Biosciences, Little Chalfont, UK). RT was then performed. For PCR amplification, XIAP primer sequences and internal reference sequences (GAPDH) were identified (Table I ). Amplification conditions used were: Pre-degenerated under 94°C for 2 min, degenerated under 94°C for 30 sec, annealed under 55°C for 45 sec, extended under 72°C for 30 sec, for a total 32 cycles, and then extended under 72°C for 10 min. This was followed by agarose gel electrophoresis on the amplification products. The ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) was used to analyze the gray level of the bands after electrophoresis, and the gene expression levels were calculated by the ratio of target gene to the level of the internal amplification product. The experiment was repeated three times.
4
Genotyping and Identification of Arcobacter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From each positive sample, eight small, colourless or beige to off-white, translucent colonies were picked, streaked to purity, and confirmed as presumptive arcobacters on the basis of their response to phenotypic tests (i.e., gram negative slightly curved rods that were positive for oxidase and motility). All isolates were genotyped using the ERIC-PCR technique, using the Houf et al. [17 (link)] protocol for Arcobacter. DNA was extracted as described above and the concentration of each DNA template was determined using the GeneQuant pro (Amersham Biosciences, Cambridge, England) at A260 and adjusted to 25 ng mL−1. Gel images were saved as TIFF files, normalized with the 100 bp DNA Ladder (Invitrogen), and further analysed by Bionumerics software, version 6.5 (Applied Maths, Belgium). Patterns with one or more different bands were considered different genotypes [17 (link)].
All strains (1 representative of each genotype) were finally identified using in parallel two molecular identification methods, the m-PCR described above for the direct detection [19 (link)] and the 16S rDNA-RFLP [1 , 20 (link)]. When identification was not possible with these methods or discordant results were obtained, the rpoB and/or 16S rRNA genes were sequenced using primers and conditions previously described [1 ].
All strains (1 representative of each genotype) were finally identified using in parallel two molecular identification methods, the m-PCR described above for the direct detection [19 (link)] and the 16S rDNA-RFLP [1 , 20 (link)]. When identification was not possible with these methods or discordant results were obtained, the rpoB and/or 16S rRNA genes were sequenced using primers and conditions previously described [1 ].
5
Conjugation of NET-1 siRNA to Avidin-Coated SMBs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SMBs were conjugated with 400 µL avidin (10 mg/mL, Sigma, USA) by incubation at 25°C. Subsequently, the SMBs were washed with PBS at 200 × g and resuspended in PBS. The avidinylated SMBs and biotinylated NET-1 siRNA were gently agitated and stored overnight at 4°C in order to allow the formation of the siRNA-SMB complexes. Then, the complexes were washed three times with PBS. The particle size and Zeta potential were measured by t a Zetasizer 3000HS (Malvern Zetasizer Nano ZS; USA). The complexes were observed by confocal laser scanning microscopy (Olympus, Japan).
The nucleic acid molecules amount of NET-1 siRNA conjugated to siRNA-SMBs complexes was determined by the absorbance of 260 nm using Gene Quant pro (Amersham Biosciences, UK).
The nucleic acid molecules amount of NET-1 siRNA conjugated to siRNA-SMBs complexes was determined by the absorbance of 260 nm using Gene Quant pro (Amersham Biosciences, UK).
6
RNA Extraction and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from each segment of the oviduct (the infundibulum, magnum, isthmus, uterus and vagina), and from the cultured vaginal tissues using Sepasol RNA I Super (Nacalai Tesque Inc., Kyoto, Japan), and dissolved in TE buffer (10 mM Tris, pH 8.0, with 1 mM EDTA). They were mixed with 1U of RQ1 RNase-free DNase (Promega Co., Madison, WI, USA), and incubated in a PTC-100 programmable thermal controller (MJ Research Inc., Waltham, MA, USA) at 37°C for 45 min and 65°C for 10 min. Concentration of RNA in each sample was measured using Gene Quant Pro (Amersham Pharmacia Biotech, Cambridge, UK). The RNA samples were then reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan). The reaction mixture (10 µL) contained 1 µg of total RNA, 1×RT buffer, 1 mM dNTP mixture, 20U RNase inhibitor, 0.5 µg of oligo (dT) 20 primer, and 50U ReverTra Ace. Reverse transcription was performed at 42°C for 30 min, followed by heat inactivation for at 99°C 5 min using the PTC-100.
7
Quantifying Superoxide and Lipid Peroxidation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amount of superoxide anion (O2·−) in the atrium was measured as described previously18 (link). Electron spin resonance spectroscopy was used to examine intracellular O2·− production with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5- tetramethylpyrrolidine hydrochloride (CMH; Alexis Corp). Briefly, freshly isolated atrial tissues was incubated with deferoxamine-chelated Krebs-HEPES solution containing CMH (0.5 mmol/L), deferoxamine (25 μmol/L), and DETC (5 μmol/L) for 90 minutes at 37 °C. Then, samples were transferred into 1-mL syringes filled with Krebs-HEPES solution and frozen in liquid nitrogen. Samples were scanned with a Bruker EMX spectrometer. Analyses of the spectra peak height were used to quantify the amount of O2·− produced by the tissue. The amount of malondialdehyde (MDA) produced from the atrium was examined by thiobarbituric acid (TBA) assay with an MDA Detection Kit (Nanjing Jiancheng Bioengineering Institute, China). In brief, stored atrium samples were weighed and 5% tissue homogenate was obtained on ice in isotonic Na chloride. 0.1 ml tissue homogenate was processed according to the manufacturer’s instructions and detected spectrophotometrically at 532 nm by an ultraviolet spectrophotometer (GeneQuant pro, Amersham Biosciences, England).
8
RNA Extraction and Reverse Transcription from Liver Slices
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 6 h of cultivation, total RNA was extracted from liver slices by Sepasol (Nacalai Tesque Inc., Kyoto, Japan). The extracted RNA was dissolved in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). For each sample, RNA concentration was estimated using Gene Quant Pro (Amersham Pharmacia Biotech, Cambridge, UK). The RNA was reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan). The reaction mixture (10 µL) contained 1 µg of total RNA, 1× RT buffer, 1 mM dNTP mixture, 20 U RNase inhibitor, 0.5 µg of oligo (dT) 20 primer, and 50 U ReverTra Ace. Reverse transcription was carried out at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using an Applied Biosystems 2720 thermocycler (Applied Biosystems, Foster City, CA, USA)
9
Quantifying Hsp27 mRNA Expression in Mo-DC and Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mRNA from the Mo-DC and tumor breast cancer cells was isolated using Illustra RNAspin Mini Isolation Kit (GE Healthcare, Piscataway, USA). The concentration of total mRNA was determined by measuring the absorbance at 260 nm using a GeneQuant pro (Amersham Biosciences, Cambridge, England). Reverse transcription was carried out with a real-time quantitative polymerase chain reaction, which contains DNAse I to avoid DNA contamination (Promega, Madison, EUA). mRNA Hsp27 expression was analyzed using Power SYBR Green master mix (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions. The fold change in target gene expression between the various groups was determined using the comparative Ct (2−ΔΔCt) method, after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin expression as an internal reference.
10
RNA Extraction and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cultured follicular theca tissues in experiments 1 and 2 using Sepasol RNA I Super (Nacalai Tesque Inc., Kyoto, Japan) and a Polytron homogenizer (Polytron PT1200ci Kinematica AG, Switzerland), and then dissolved in TE buffer (10 mM Tris, pH8.0, with 1 mM EDTA). The concentration of RNA in each sample was measured using Gene Quant Pro (Amersham PharmaciaBiotech, Cambridge, UK). RNA samples were mixed with RQ1 RNase-free DNase (Promega Co., Madison, WI, USA) in a 10 µL reaction mixture (10 µg of total RNA, 1× DNase buffer, and 1U DNase) and incubated in a PTC-100 programmable thermal controller (MJ Research, Inc., Waltham, MA, USA) at 37°C for 45 min and at 65°C for 10 min. The RNA samples were reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan), according to the manufacturer's instructions. Reverse transcription (RT) was performed at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min, using the PTC-100 programmable thermal controller (MJ Research, Inc.). The reaction mixture (10 µL) contained 1 µg of purified RNA, 1× RT buffer, 1 mM dNTP mixture, 20U RNase inhibitor, 0.5 mM oligo (dT) 20 primer, and 50 U ReverTra Ace.
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!