Total RNA was extracted from the livers of SHR-CRP transgenic and SHR control rats (N = 4 per group) treated with metformin or with placebo. The quality and concentration of RNA were determined using a NanoDrop 2000 spectrophometer (Thermo Scientific). RNA integrity was analyzed in an Agilent Bioanalyzer 2100. We only included samples judged to have an intact RNA profile. The Affymetrix GeneChip® Rat Gene 1.0 ST Array System was used for the microarray analysis following the standard protocol: 100 ng RNA was amplified with an Ambion WT Expression Kit (Applied Biosystems), 5.5 μg single-stranded cDNA was labeled and fragmented with GeneChip WT Terminal Labeling and Hybridization (Affymetrix) and hybridized on the chip according to the manufacturer’s procedure. The analysis was performed in three replicates. The transcription data were MIAME-compliant and deposited in the ArrayExpress database (ID # E-MTAB-3791).
Nanodrop 2000 spectrophometer
Manufactured by Thermo Fisher Scientific
The NanoDrop 2000 spectrophotometer is a compact and versatile instrument designed for the quantification of small-volume samples. It utilizes a patented sample retention system that requires only 1-2 microliters of sample to perform measurements. The NanoDrop 2000 can measure the concentration and purity of DNA, RNA, and protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Ambion wt expression kit, by Thermo Fisher Scientific (1 mentions)
Genechip rat gene 1.0 st array system, by Thermo Fisher Scientific (1 mentions)
Genechip wt terminal labeling and hybridization, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Taqman expression assay, by Thermo Fisher Scientific (1 mentions)
Rotor gene q machine, by Qiagen (1 mentions)
Kapa probe fast universal qpcr master mix, by Roche (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Flashgel dna cassette, by Lonza (1 mentions)
Neb monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Phusion polymerase, by New England Biolabs (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1 treatment, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
5 protocols using nanodrop 2000 spectrophometer
1
Transcriptional Profiling of Liver in SHR Rats
2
Recombinant Protein Purification Workflow
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Cell cultures harboring expression vectors were grown in lysogeny broth at 37 °C and 180 rpm until cells reached mid-log phase (OD600 0.4–0.6), at which point protein production was induced by addition of 0.2 mM isopropyl-β-d -1-thiogalactopyranoside, and cells cultured overnight (16 °C and 180 rpm). The cells were harvested by centrifugation and lysed by sonication. The resulting protein containing crude lysate was purified using immobilized metal ion affinity chromatography as previously described37 (link). Purified protein was concentrated and buffer exchanged (BeCE15A in 50 mM Tris pH 8.0 + 100 mM NaCl; BeRex8A in sodium phosphate pH 6.5 + 100 mM NaCl; and BeCE15A-Rex8A in 50 mM Tris pH 8.0 + 250 mM NaCl + 5% w/v glycerol) using 10 kDa cut-off centrifugal filter units (Amicon Ultra-15, Merck-Millipore) and imidazole concentrations were reduced to < 1 mM. Sodium dodecyl sulfate polyacrylamide gel electrophoresis using Mini-PROTEAN TGX Stain-Free Gels (BIO-RAD) was used to verify molecular weight and protein purity. Protein concentrations were determined using a Nanodrop 2000 Spectrophometer (Thermo Fisher Scientific) using extinction coefficients and molecular weights predicted by Benchling.
3
RT-qPCR Analysis of Immune Genes
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Late third-instar larvae were washed and dipped in 70% ethanol followed by rinsing with water. Total RNA extraction was performed as previously described (Arefin et al. 2014 (link)). RNA quality and concentration was determined with NanoDrop 2000 spectrophometer (Thermo Scientific). A total of 1000 ng of total RNA was used to convert cDNA using SuperScript III Reverse Transcriptase (Invitrogen) and oligo(dT) (20-mer). RT-qPCR was performed using KAPA PROBE FAST Universal qPCR Master Mix (Kapa Biosystems) and TaqMan Expression Assays (Applied Biosystems) for Drs and Cecropin A1. Diptericin A1 was synthesized by the custom TaqMan Expression Assays (Applied Biosystems) (Arefin et al. 2015 (link); Dantoft et al. 2013 (link)). A Rotor-gene Q machine (Qiagen) was used to run qPCR. Each sample was run in triplicate. Relative mRNA expression levels were was analyzed by normalizing to the reference gene rpl32 and compared to the control genotype. The data were then transformed to Log2 fold changes and presented as mean ± SDs of the mean of at least three independent biological replicates. In the uninfected wounded situation, two independent biological samples were analyzed and both show a similar pattern.
4
Restriction Digest and PCR Amplification
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Fragments 1 and 2 were prepared and transcribed at separate times to prevent any cross contamination.
For both fragments, a double restriction digest was performed with KasI (New England Biolabs, Ipswich, MA, catalog #R0544L) and NruI (New England Biolabs, catalog #R0192L) to cut the desired construct from the backbone plasmid. After digestion, the DNA size was confirmed on a FlashGel DNA Cassette (Lonza, Walkersville, MD, catalog # 57,023), and amplified with M13 F and R primers and Phusion polymerase (New England Biolabs, catalog #M0531S). The PCR conditions were as follows: in a 50 μL reaction, 25 μL of 2x Phusion Master Mix was combined with 2.5 μL each of 10 μM M13 F primer and M13 R primer, 2 μL of linearized DNA and 18 μL of molecular biology grade water. Thermal cycling conditions for this reaction were: 98 °C for 30 s, then 35 cycles of 98 °C for 7 s, 55 °C for 20 s, 72 °C for 60 s; and 10 min at 72 °C.
After PCR amplification, the size and purity of the expected PCR product was confirmed on a FlashGel and purified with the NEB Monarch PCR & DNA Cleanup Kit (New England Biolabs, catalog #T1030L) according to the manufacturer's instructions. After purification, the DNA concentration was measured with the Nanodrop 2000 Spectrophometer (Thermo Fisher, Waltham, MA, catalog #ND2000) prior to RNA transcription.
For both fragments, a double restriction digest was performed with KasI (New England Biolabs, Ipswich, MA, catalog #R0544L) and NruI (New England Biolabs, catalog #R0192L) to cut the desired construct from the backbone plasmid. After digestion, the DNA size was confirmed on a FlashGel DNA Cassette (Lonza, Walkersville, MD, catalog # 57,023), and amplified with M13 F and R primers and Phusion polymerase (New England Biolabs, catalog #M0531S). The PCR conditions were as follows: in a 50 μL reaction, 25 μL of 2x Phusion Master Mix was combined with 2.5 μL each of 10 μM M13 F primer and M13 R primer, 2 μL of linearized DNA and 18 μL of molecular biology grade water. Thermal cycling conditions for this reaction were: 98 °C for 30 s, then 35 cycles of 98 °C for 7 s, 55 °C for 20 s, 72 °C for 60 s; and 10 min at 72 °C.
After PCR amplification, the size and purity of the expected PCR product was confirmed on a FlashGel and purified with the NEB Monarch PCR & DNA Cleanup Kit (New England Biolabs, catalog #T1030L) according to the manufacturer's instructions. After purification, the DNA concentration was measured with the Nanodrop 2000 Spectrophometer (Thermo Fisher, Waltham, MA, catalog #ND2000) prior to RNA transcription.
5
In Vitro RNA Transcription and Purification
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RNA fragments were transcribed using a MEGAscript T7 kit (Thermo Fisher, catalog # AM1334) according to the manufacturer's instructions using approximately 200 ng of the PCR product. The transcription reaction was incubated for 2 h at 37 °C. After 2 h, 1 μL of Turbo DNase was added and the mixture was incubated for 15 min at 37 °C. The RNA was purified with the RNeasy Mini Kit (QIAGEN, Germantown, MD, catalog # 74104) with on column DNase I treatment (QIAGEN, catalog #79254) per the manufacturer's instructions. At the end of the purification protocol, the RNA was eluted from the column with two successive elutions using 50 μL of molecular biology grade water. The eluted RNA was pooled, and the bulk concentration was measured with the NanoDrop 2000 Spectrophometer (Thermo Fisher).
After purification, the RNA fragment size was confirmed using both an RNA FlashGel Cassette (Lonza, catalog # 57027) and the 2100 Bioanalyzer System with an RNA 6000 Nano Kit (Agilent, Santa Clara, CA, catalog #5067–1511) according to the manufacturer's instructions.
After purification, the RNA fragment size was confirmed using both an RNA FlashGel Cassette (Lonza, catalog # 57027) and the 2100 Bioanalyzer System with an RNA 6000 Nano Kit (Agilent, Santa Clara, CA, catalog #5067–1511) according to the manufacturer's instructions.
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