Total RNA from liver specimens was isolated at E20, PW3, and PW40 using the TRIzol reagent (Invitrogen), and quantified with the Nanovue instrument (GE Healthcare, Buckinghamshire, England). The RNA strand (2 µg) was reverse-transcribed into cDNA using the RNA PCR kit (TaKaRa, Dalian, China). The expression of the cDNAs of interest was measured by quantitative real-time PCR using the SYBR Premix Ex Taq kit (TaKaRa) and a Lightcycler detector (Roche, Basel, Switzerland), and was normalized with β-actin used for housekeeping. The results were expressed in arbitrary units relative to the mean value of the control group. The specific primer sets used are listed in Table 1 .
Nanovue instrument
Manufactured by GE Healthcare
Sourced in United Kingdom
The NanoVue instrument is a high-precision spectrophotometer designed for analyzing small sample volumes. It provides accurate measurements of nucleic acid and protein concentrations in a compact and user-friendly device.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler detector, by Roche (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Rna pcr kit, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Pcr clean up kit, by Macherey-Nagel (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using nanovue instrument
1
Liver RNA Expression Analysis
2
Phylogenetic Analysis of Bacterial Isolates
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After classification, one isolate per group was selected. The 16S rRNA-encoding gene was amplified by PCR as described above, purified with a PCR clean-up kit (Macherey-Nagel) and the concentration was evaluated with NanoVue instrument (GE Healthcare).
The DNA amplified and purified was diluted to a final concentration of 10 μg/mL in a volume of 15 μL and then sequenced. The sequencing was carried out by Eurofins MWG Operon Company. The sequences obtained were searched against NCBI, EMBL and DDJB databases using BLASTN 2.0.5. The GenBank accession number of the 16S rRNA-encoding gene sequences are reported below:
group A: MZ054377, group B: MZ054378, group C: MZ054379, group D: MZ054380, group E: MZ054381, group F: MZ054382, group G: MZ054383, group H: MZ054384, group I: MZ054385, group K: MZ054386.
The identity ≥ 99% with e-value of 0.0 was found for each species.
Phylogenetic trees were constructed by neighbour-joining methods using Molecular Evolutionary Genetics Analysis (MEGA) version 6.06 [60 (link)]. The topology of the tree was evaluated by means of bootstrap analysis based on 1000 replicates.
The DNA amplified and purified was diluted to a final concentration of 10 μg/mL in a volume of 15 μL and then sequenced. The sequencing was carried out by Eurofins MWG Operon Company. The sequences obtained were searched against NCBI, EMBL and DDJB databases using BLASTN 2.0.5. The GenBank accession number of the 16S rRNA-encoding gene sequences are reported below:
group A: MZ054377, group B: MZ054378, group C: MZ054379, group D: MZ054380, group E: MZ054381, group F: MZ054382, group G: MZ054383, group H: MZ054384, group I: MZ054385, group K: MZ054386.
The identity ≥ 99% with e-value of 0.0 was found for each species.
Phylogenetic trees were constructed by neighbour-joining methods using Molecular Evolutionary Genetics Analysis (MEGA) version 6.06 [60 (link)]. The topology of the tree was evaluated by means of bootstrap analysis based on 1000 replicates.
3
Isolation and Quantification of Cellular RNA
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Total cellular RNA was isolated by extraction with Trizol (Invitrogen). RNA was purified by phenol–chloroform extraction, followed by ethanol precipitation. RNA samples were treated with DNase I (Invitrogen) for 15 min at 37°C, and the enzymatic reaction was quenched by the addition of DNase-inactivation resin. RNA concentrations and purities were assessed with a NanoVue instrument (GE Healthcare Life Sciences). Purified cellular RNA (∼1 μg) was used in the reverse transcription reaction along with random hexamers from the SuperScript III Reverse Transcriptase kit (Invitrogen). A 1-μl solution of the ensuing cDNAs was used in qPCR reactions performed with the PerfeCTa SYBR Green FastMix cocktail (Quantabio). Amplified cDNAs were evaluated with an ABI Prism 7200 sequence detector (Perkin Elmer) using the primers listed in Supplementary Table S1. Data were analyzed by the comparative CT method (35 ) using the actin gene as an endogenous standard.
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