Total RNA samples were isolated from Synechocystis and its mutants using ZR Fungal/Bacterial RNA MiniPrep kit (Zymo Research). The quantity and quality of the isolated RNAs were determined using Nanodrop. Synthesis of cDNAs was performed with random primers following the manufacturer’s protocol (Thermo Scientific). The synthesized cDNAs were used as templates for qPCR to detect the transcription of the integrated PPTase genes, while the isolated RNA samples themselves were used as the templates of PCR reactions to detect any residual genomic DNAs using primers listed in Table S1 . The student’s t-test analysis was applied to determine significance difference between the samples, and a P-value < 0.05 was considered to be statistically significant.
Zr fungal bacterial rna miniprep kit
Manufactured by Zymo Research
Sourced in United States, Denmark
The ZR Fungal/Bacterial RNA MiniPrep kit is a laboratory product designed for the purification of total RNA from a variety of fungal and bacterial samples. The kit utilizes a simple and efficient method to isolate high-quality RNA for downstream applications such as RT-PCR, Northern blotting, and microarray analysis.
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Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
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15 protocols using zr fungal bacterial rna miniprep kit
1
Synechocystis mutant RNA analysis
2
Pectin Impact on Bacterial Transcriptome
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Total RNA was isolated from cells grown in TSB without pectin and in TSB supplemented with 0.4% w/v of either PGA, apple pectin, or RG-I from soy at 12.75 h after inoculation with the ZR Fungal/Bacterial RNA Miniprep kit (Zymo Research, Irvine, CA). Ten µg of RNA was treated with DNase using the TURBO DNA-free Kit (Thermo Fisher, Waltham, MA). RNA was used as a PCR template to confirm that no genomic DNA was remaining. One microgram of each sample was reverse transcribed with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and diluted 1:10 with nuclease-free water. Two microliter of diluted cDNA was used as template in 20-µL qPCRs with Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA) and run on a StepOne Plus instrument (Applied Biosystems, Foster City, CA). All conditions were repeated with four biological replicates. Oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, IA).
Six potential reference genes (ftsZ, rpoD, era, adk, gyrA, and gap homologues) were evaluated and ranked using the geNorm algorithm [45 (link)] and two most stable genes, ftsZ and rpoD, were selected as endogenous control genes. Quantification thresholds and Cq values were calculated by the StepOne Plus software. Fold change in expression was calculated using the 2−∆∆Cq method [28 (link)].
Six potential reference genes (ftsZ, rpoD, era, adk, gyrA, and gap homologues) were evaluated and ranked using the geNorm algorithm [45 (link)] and two most stable genes, ftsZ and rpoD, were selected as endogenous control genes. Quantification thresholds and Cq values were calculated by the StepOne Plus software. Fold change in expression was calculated using the 2−∆∆Cq method [28 (link)].
3
Quantitative RT-PCR Analysis of E. festucae
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Total RNA was isolated from frozen mycelium of the E. festucae Rose City isolate and from leaf sheath tissue of the Rose City isolate-infected plant tissue. The tissues were homogenized in a HT Mini bead beater (Ops Diagnostics, Lebanon, NJ, USA) and RNA extracted using the ZR Fungal/Bacterial RNA MiniPrep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. A TURBO DNA-free kit (Invitrogen, Carlsbad, CA, USA) was used to remove any genomic DNA in the RNA samples. cDNA synthesis was performed on 1.5 μg RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on a StepOnePlus RT-PCR system (Applied Biosystems) with three technical replicates per sample. Efe-afpA transcripts were normalized to the expression levels of elongation factor 2 (EfM3.021210) and 40S ribosomal protein S22 (EfM3.016650) using primers described by Chujo and Scott [25 (link)] and the 2−ΔΔCt method as described by Livak and Schmittgen [26 (link)]. Primer sequences used for quantitative RT-PCR are given in Table S1 .
4
Quantitative Analysis of Bacterial Fimbrial Gene Expression
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Total RNA was extracted and purified from bacterial strains cultured in static broth with the ZR Fungal/Bacterial RNA Miniprep™ kit (Zymo Research, Irvine, CA) according to the manufacturer's instructions. To remove the residual DNA, all the extractions were treated with RNase‐free DNase I (ThemoFisher Scientific, Waltham, MS). The purified RNA (0.1 μg) was converted to cDNA using iScript™ cDNA Synthesis kit (Bio‐Rad Laboratories, Hercules, CA), and transcribed into mRNA using iTaq™ universal SYBR® Green supermix (Bio‐Rad Laboratories). Transcription of fimA, fimY, fimZ, and 16s rRNA as an internal control were detected by quantitative RT‐PCR. Cycling conditions were performed using CFX Connect™ Real‐Time System (Bio‐Rad Laboratories) as follows: 95°C for 3 min followed by 49 cycles of 95°C for 10 s and 60°C for 30 s. Melting curves and nontemplate controls were included to detect any primer dimerization or other artifacts. The mRNA transcript levels were analyzed by the software Bio‐Rad CFX Manager 3.1 (Bio‐Rad Laboratories).
5
RNA Extraction and Quantification
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For RNA extraction, the “ZR Fungal/Bacterial RNA MiniPrep” Kit (Zymo Research) and the corresponding protocol was used, using lysozyme treatment (at 1 μg/ml for 10 min) of the cells instead of bead beating for cell lysis. RNA was converted to cDNA by the “iScript Select cDNA Synthesis” Kit (BIO-RAD Laboratories). qPCR was conducted with “SsoAdvanced Universal SYBR Green Supermix” (BIO-RAD Laboratories). The relative quantification of the transcripts was performed with the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)).
6
Transcriptional Analysis of V. fischeri
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RNA was extracted from wild-type or mutant V. fischeri strains following incubation for 5 or 12 h at 24°C on LBS agar plates supplemented with the appropriate antibiotic and IPTG when indicated. RNA extractions were performed according to the ZR Fungal/Bacterial RNA Miniprep kit (Zymo Research, Orange, CA). Transcriptional analysis was performed according to the ONETAQ One-Step RT-PCR kit (NEB, Ipswich, MA), and similar concentrations of DNA, RNA, or cDNA were added to PCR mixtures within experiments. All RT-PCR experiments were performed on two separate occasions with RNA derived from separate extractions, each with similar results.
7
RNA Extraction from Algal Cultures
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For RNA extraction, 200 ml samples were taken at the end of the exponential growth phase (Additional file 1 : Figure S1). The samples were taken at the middle of the light period and exactly 12 h later at the middle of the dark period) and kept in the dark in aluminum foil until cell lysis. Per sample, 200 ml of cell culture was centrifuged in 4 aliquots of 50 ml in TubeSpin® Bioreactor50 tubes (TPP) at 5000 G. Supernatant was carefully discarded and remaining medium was removed from the inside of the tube to prevent the high salinity from interfering with the RNA isolation procedure. RNA was extracted from each of the 8 pellets using the ZR Fungal/Bacterial RNA MiniPrep kit (Zymo Research, Irvine, USA) following the manufactures recommendations and eluted in 100 μl of elution buffer. After RNA extraction the 4 samples were pooled resulting in one night and one day sample with an average RNA yield of 300 ng/μl and a total yield of 120 μg. Contaminating DNA fractions were removed using TURBO DNA-free Kit (Ambion, USA) following the manufacturers recommendations. The yield and quality of the resulting RNA fractions were checked on an RNA6000 chip using the 2100 Bioanalyzer system (Agilent, Santa Clara, USA).
8
qRT-PCR Analysis of degP Expression
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Total RNA was isolated from the biofilm cell pellets using a ZR Fungal/Bacterial RNA MiniPrep™ kit (Zymo Research, Orange, CA, USA) and quantified by a UV 1600-PC spectrophotometer (VWR, Darmstadt, Germany) reading OD at 260 nm and 280 nm. 50 ng of total RNA was mixed with an iTaqTM Universal SYBR green one-step kit (Bio-Rad, Hercules, CA, USA) by following the manufacturer’s protocol, and qRT-PCR was performed using degP and rrsG RT-PCR primers (Supplementary Table 7 ) in a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The expression levels of degP and rrsG, a housekeeping gene of 16 S ribosomal RNA of rrnG operon, were monitored, and the CT value was compared to analyze the degP expression levels in different samples.
9
Fungal Transcriptome Analysis Protocol
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After 8 weeks of incubation, the loads of mycelia from each treated sample bottle were separated from the growth media by filtrating out the spent media. Using a sterile spatula, duplicate samples from each mycelial load were removed, weighed in cryogenic tubes, frozen in liquid nitrogen, and then crushed with a micro pestle before transfer to a bead bashing tube, followed by vortexing for 10 minutes. Using the ZR Fungal/Bacterial RNA Mini Prep kit (Zymo Research Corporation, Irvine, CA, USA), RNA was extracted according to the manufacturer's instructions and sent to the Genome Core facility at the Waksman Institute in Rutgers University for quality check, preparation, and sequencing. An Illumina NextSeq 500 instrument was used to yield single-end 1 × 75 bp reads. Sequencing reads without adapter sequences were deposited in the NCBI SRA database under accession number PRJNA523070.
10
Fungal/Bacterial RNA Extraction and cDNA Synthesis
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Total RNA was extracted using the ZR Fungal/Bacterial RNA MiniPrep™ kit according to the manufacturer's instructions (Zymo Research, Nordic Biosite, Copenhagen, Denmark). Contaminating DNA was removed using the DNA‐free™ DNA removal kit (Fisher Scientific) according to the manufacturer's instructions. RNA purity and concentration were determined using Qubit® RNA HR Assay Kit (Fisher Scientific) and RNA quality was assessed by agarose gel electrophoresis. First‐strand cDNA synthesis was performed using ProtoScript® first‐strand synthesis kit (NEB) according to the manufacturer's instructions.
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