Three pools of 5 larvae from each treatment group were used for RNA isolation. Following imaging at 15 dpf, RNA Later (Fisher Scientific) was added to 5 larvae per treatment group and stored at −80°C until RNA isolation. Though individually housed from 5–15 dpf, larvae were randomized from the initial exposure vials to minimize batch effects. RNA was isolated using the GeneJET RNA purification Kit (Fischer Scientific, Waltham, MA, USA). The isolation protocol followed was developed for use of whole-tissue purification. The concentration of RNA and the purity of samples were analyzed using a μLITE spectrophotometer (BioDrop, Cambridge, UK), and all samples had quality A260/A280 ratios ranging from 1.8–2.1. After quantifying RNA, 500 ng RNA was reverse transcribed into cDNA with an iScript cDNA Synthesis Kit (Bio-Rad). These samples were diluted to a concentration of 0.25 ng/μl of cDNA in nuclease-free water and stored at −20°C until use.
μlite spectrophotometer
Manufactured by Harvard Bioscience
Sourced in United Kingdom
The μLITE spectrophotometer is a compact, high-performance device designed for precise measurement of absorbance in various biological and chemical applications. It features a dual-beam optical system and a wavelength range from 190 to 1100 nm, allowing for accurate analysis of samples.
Automatically generated - may contain errors
Lab products found in correlation
Genejet rna purification kit, by Thermo Fisher Scientific (2 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Iscript reaction mix kit, by Bio-Rad (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
2 mercaptoethanol, by MP Biomedicals (1 mentions)
Lightcycler 96 analyzer, by Roche (1 mentions)
4 protocols using μlite spectrophotometer
1
RNA Isolation and cDNA Synthesis
2
Zebrafish RNA Extraction and RT-qPCR Analysis
3
RNA Extraction and Gene Expression Analysis
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Total RNA was extracted from the third mature leaf in three biological replicates by the CTAB method (Doyle and Doyle, 1991 ) with minor modifications. The concentration and quality of RNA were determined using BioDrop μLite spectrophotometer and integrity was assessed by agarose gel electrophoresis. RNA samples were treated with DNase I and reverse transcription was performed using the MMLV-RT kit (Biolabmix, Russia). The efficiency of DNaseI treatment and reverse transcription were tested by agarose gel electrophoresis and by qRT-PCR. The results of this verification were evaluated by the presence/absence of a PCR product in RNA samples before and after DNaseI treatment, and by observing the size of PCR fragments in RNA samples before treatment and its cDNA synthesis. Only those samples that confirmed the absence of genomic DNA contamination were included in further analysis of gene expression. Actin (F: 5′-CCATCACCAGAATCCAAGAC-3′; R 5′-GAACCCGAAGGCGAATAGG-3′) (Hao et al., 2014 (link)) was taken as a reference gene and results were quantified using a Light Cycler 96 analyzer (Roche, Japan). The relative gene expression level was calculated by the Livak and Schmittgen (2001 (link)) using the following algorithm: 2−ΔΔCq, where:
4
Fecal DNA Extraction with CTAB
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A modified version of the CTAB protocol was used for DNA extractions due to the likely presence of PCR inhibitors in the fecal samples (Ramón‐Laca et al., 2015 ). Each swab was placed in a lysis mix of CTAB and proteinase k (20 mg/ml) and left overnight at 56°C. This was followed by the addition of RNaseA and incubation of the mixture at 37°C for 30 min. DNA was extracted using chloroform–isoamyl alcohol and then precipitated using isopropanol and ethanol. A negative control consisting of purified water was included in each set of extractions. DNA concentration was quantified using a BioDrop μLITE spectrophotometer.
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