Prior to DNA extraction, the aliquots were homogenised a second time, to avoid variability caused by changes in the microenvironments after 48 hours of storage. For the first five samples, three different commercially available stool DNA extraction kits were compared: PSP® Spin Stool DNA Kit (Stratec Biomedical, Germany), ZymoBIOMICS DNA Miniprep Kit (Zymo Research, USA) and QIAamp PowerFecal DNA Isolation Kit (Qiagen, formerly moBio, Germany). The workflow is shown in Fig 1 . Briefly, for each kit, DNA was extracted from 200 mg aliquots of stool from each of the four storage conditions, according to the manufacturer’s instructions. Aliquots frozen at -80°C were not thawed prior to extraction. DNA purity (A260/A280 and A260/A230), and yield (ng/μL) were determined using the BioDrop μLite spectrophotometer (BioDrop, UK) and by Qubit Fluorometric Quantitation (Invitrogen, USA) at the Central Analytical Facility (CAF) of Stellenbosch University. We used the Kruskal-Wallis test [13 ] for non-normally distributed data to determine whether the different extraction kits or storage methods influenced the DNA yield significantly (p < 0.05). DNA degradation was assessed by gel electrophoresis. The remaining five samples were subjected to the same storage condition comparisons as described above and DNA was extracted with the kit that showed the best performance.
Biodrop μlite spectrophotometer
Manufactured by Harvard Bioscience
Sourced in United Kingdom
The BioDrop μLite spectrophotometer is a compact, single-beam UV-Vis spectrophotometer designed for the measurement of nucleic acid and protein concentrations. It features a wavelength range of 198 to 830 nm and can accommodate sample volumes as low as 0.5 μL.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometric quantitation, by Thermo Fisher Scientific (1 mentions)
Qiaamp powerfecal dna isolation kit, by Qiagen (1 mentions)
Zymobiomics dna miniprep kit, by Zymo Research (1 mentions)
Psp spin stool dna kit, by Stratec (1 mentions)
Applied biosystems high capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Maxima h minus double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Thermo cycler, by Merck Group (1 mentions)
Minelute gel extraction kit, by Qiagen (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Hiseq platform, by Illumina (1 mentions)
6 protocols using biodrop μlite spectrophotometer
1
Stool DNA Extraction Kit Comparison
2
Gene Expression Analysis of Renal Fibrosis
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Cells were cultured in 6-well plates (105 cells/well) and treated with ASE (10 μg/mL), p-CS (40 μg/mL) or IS (63 μg/mL), followed by a 24 h incubation period. The cells were then washed with ice-cold PBS and RNA extraction was performed with Trizol® (1 mL/well). Total RNA was quantified using a BioDropμLITE spectrophotometer (BioDrop, UK). In a further step, RNA was converted to cDNA (complementary DNA) and quantified by real time qPCR (Applied Biosystems High-Capacity RNA-to-cDNA Kit, Life Technologies, MA, USA). Reaction parameters were considered as follows: 60 min at 37 °C; 5 min at 95 °C; cooling at 4 °C (thermocycler Veriti 96 Well Thermal Cycler, Applied Biosystems, MA, USA). TaqMan® gene expression assay (Applied Biosystems, MA, USA) for Homo sapiens (Hs) was used to detect the following parameters: Collagen 1α (Col-1α; Hs01103890_m1), transforming growth factor β1 (TGF-β1; Hs00608187_m1), connective tissue growth factor (CTGF; Hs00170014_m1), α-smooth muscle actin (α-SMA; Hs01566408_m1), and glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH; Hs01548420_m1) mRNA expression. Quantitative real time PCR was performed using 7500 Fast Real-Time PCR System (Applied Biosystems, MA, USA). Primers sequences are listed in Supplementary Table 2 .
3
RNA Extraction from Fruit Tissues
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Total RNA was extracted using a modified CTAB method [16 ]. In the first in-field sampling (2018), we extracted RNA from fruit/pedicel tissues (pooled from four biological replicates) for RNA-Seq and metabolomics analyses. In the next two sampling of ex vivo experiments (2019–2020), RNA was extracted from each tissue of tree subunits (pooled from eight biological replicates) for qRT-PCR and microscopic analyses. The concentration of RNA was measured using BioDrop μLITE spectrophotometer (Cambridge, UK).
4
dsRNA Extraction and cDNA Synthesis
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The dsRNA was extracted using a protocol previously described by Nyaga et al.[35 (link)], and purified using the Qiagen MinElute gel extraction kit (Qiagen, Hilden, Germany). The quality of the purified dsRNA was thereafter verified by 1% agarose gel electrophoresis prior to quantification using a BioDrop-μLITE spectrophotometer (Biodrop, Cambridge, UK). For the cDNA synthesis, the Maxima H Minus Double-Stranded cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) was used in accordance to manufacturer’s instructions with minor modifications. The modification included the denaturation of the dsRNA at 95 °C as a replacement for 65 °C for five minutes prior to synthesis of the first strand for 2 h at 50 °C in a thermocycler (Merck, Darmstadt, Germany).
5
Developmental Gene Expression in Zebrafish Embryos
6
RNA-seq Analysis of Cucumber Cotyledon
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RNA was extracted according to the method described in the RNAprep Pure Plant Kit (Tiangen, Beijing, China). The purity and concentration of RNA were detected with a BioDrop μLite spectrophotometer (BioDrop in Cambridge CB4 OFJ England). The integrity of the RNA was assessed by 1% agarose gel electrophoresis. RNA samples extracted from B21-a-2-1-2 and B21-a-2-2-2 cucumber cotyledons harvested at 0 and 6 hpi were sequenced on the Illumina Hiseq platform (Personalgene, Nanjing, China). Equal volumes of the RNA from the three biological replicate samples at each time point were mixed prior to sequencing.
Clean data was obtained by filtering out the joints and low-quality reads of raw data. Clean data was mapped to the cucumber genome database1 via HISAT2 (an updated version of TopHat2).2 The mapping was considered successful when the mismatches between the default reads and the reference genome sequences were within 2. Htseq was used to calculate the read count value mapped to each gene, and this value was considered the original level of expression of the gene. The expression was then standardized as fragments per kilobase million (FPKM). DESeq was used for differential analysis of gene expression. The screening conditions of differentially expressed genes (DEGs) were | log2FoldChange| > 1 and p-value < 0.05.
Clean data was obtained by filtering out the joints and low-quality reads of raw data. Clean data was mapped to the cucumber genome database
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