For all experiments, total RNAs were isolated using a Qiagen plant RNA mini kit or Omega EZNA Plant RNA kit (Omega Bio‐Tek, Norcross, GA, USA) following the manufacturer’s instructions, including on‐column DNAse treatment. Either 1 µg or 500 ng total RNA was used for reverse transcription using an iScript cDNA synthesis kit (Bio‐Rad). Quantitative PCR (qPCR) was done using an iQ SYBR Green Supermix (Bio‐Rad) on a Bio‐Rad CFX real‐time platform. All plant and fungal genes were normalized using M. truncatula or Rhizophagus irregularis elongation factor 1α. Milli‐Q water was used as negative control in all qPCR analyses. The primers used are shown in Table S1 .
Omega ezna plant rna kit
Manufactured by Omega Bio-Tek
Sourced in United States
The Omega EZNA Plant RNA kit is a laboratory tool designed to efficiently extract and purify high-quality total RNA from a variety of plant tissues. The kit utilizes a specialized protocol and reagents to ensure effective cell lysis, RNA binding, washing, and elution, providing a reliable method for obtaining RNA samples suitable for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Transscript all in one first strand cdna synthesis supermix for qpcr kit, by Transgene (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Plant rna mini kit, by Omega Bio-Tek (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Transstart top green qpcr supermix kit, by Transgene (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Perfectstart uni rt, by Transgene (1 mentions)
Easyscript one step gdna removal and cdna synthesis supermix, by Transgene (1 mentions)
Sybr green qpcr mix, by Sparkjade (1 mentions)
6 protocols using omega ezna plant rna kit
1
RNA Isolation and qPCR Analysis Protocol
2
Gene Expression Analysis by qPCR
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Total RNA was extracted using an OMEGA E.Z.N.A. Plant RNA Kit (Omega Bio-Tek, Norcross, GA, USA). According to the manufacturer’s instructions, the qualified RNA was reverse-transcribed into cDNA using a TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (TransGen Biotech, Beijing, China). Gene expression detection was performed with the utility of a TransStart® Top Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China) on a real-time fluorescence quantitative PCR instrument (Bio-Rad CFX96, Hercules, CA, USA), with an annealing temperature of 60 °C and a cycle number of 40. With the glyceraldehyde-3-phosphate dehydrogenase gene (FfGAPDH) as an internal reference, quantitative PCR primers were designed based on Primer-BLAST online software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome (accessed on 5 June 2017) and synthesized by Tsingke Biotechnology Co., Ltd. (Tianjin, China). The primer sequences are displayed in Table 1 . The relative expressions of the target genes were calculated using the 2−ΔΔCt method [27 (link)].
3
RNA Extraction and cDNA Synthesis
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RNA was extracted from frozen milled leaf and root sample using the omega E.Z.N.A™ Plant RNA Kit (Omega bio-tek, http://www.omegabiotek.com/ ). Quality and quantity of the RNA were assessed by measuring the A260/A280 ratio with NanoDrop 1000 spectrophotometer (Thermo-scientific, USA). The first-strand cDNA was synthesized using PrimerScript 1st Stand cDNA Synthesis Kit (Takara) from 1 μg RNA in the final volume of 15 μL.
4
Transcriptional Analysis of SA Signaling Pathway
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The samples of mycelia growing on the surface of the substrate under four treatments and the control were collected 3 h after the treatments (referring to the results of Han et al., 2022 (link)) for the determination of transcriptional levels of SA signaling pathway genes by RT-qPCR. All the samples were collected in equal mass (0.5 g) and frozen in liquid nitrogen for RNA extraction. Total RNA was extracted according to the standard method described in the instructions of the Omega E.Z.N.A. plant RNA kit (Omega Bio-Tek, USA). When the A260/A280 ratio of RNA is 2.0 to 2.1 (measured using Thermo Science NanoDrop Lite; USA) and the concentration is diluted to 500 ng/μl, it is used for cDNA synthesis. The cDNA was synthesized by reverse transcription using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit (TransGen, China). Three internal reference genes stably expressed in F. filiformis (RNB, V-ATP, and β-TUB) were used for the standardization of RT-qPCR (Yang et al., 2022 (link)). All the primers for RT-qPCR are shown in Supplementary Table S1 . The RT-qPCR was performed according to the standard method described in the instructions of PerfectStart Uni RT and qPCR Kit (TransGen Biotech, Beijing). The relative gene expression levels were determined according to the 2−ΔΔCt method.
5
Quantifying Laccase Expression in Heavy-Metal Stress
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Total RNA of samples was extracted using the Omega E.Z.N.A Plant RNA Kit (Omega Bio-Tek, Norcross, Georgia, USA) and reverse-transcribed into cDNA using Easyscript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co., Ltd., Beijing, China). To quantify the relative expression level of laccases in response to heavy-metal stress, qRT-PCR was performed using 2 × SYBR Green qPCR Mix (Shandong Sparkjade Biotechnology Co., Ltd., Qingdao, China). PvUbq2 (GenBank accession NO: HM209468) was used as reference to normalize the expression data. The primers used in this work are listed in Table S2 .
6
Quantification of Fungal MfCYP51 Expression
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A total of 16 isolates were evaluated for MfCYP51 expression, including four isolates in the following categories: no Mona insert, 311-bp insert, 376-bp insert, and 1,815-bp insert. Each isolate was grown on PDA medium overlaid with sterile cellophane as described above. Isolates cultured on PDA medium were grown for approximately 5 days at 25°C. Approximately 100 mg of mycelium for each isolate was harvested and immediately ground with a precooled mortar and pestle using liquid nitrogen. Total RNA was immediately extracted using the "difficult sample type" protocol of the Omega E.Z.N.A Plant RNA kit (Omega Bio-Tek, Norcross, GA) following the manufacturer's instructions. Residue DNA contamination was removed using the Turbo DNA-Free Kit (Thermo Fisher Scientific, Waltham, MA). Quantity and quality of RNA was assessed through NanoDrop 1000 (Thermo Fisher Scientific) and gel electrophoresis. cDNA was synthesized using 1 µg of RNA in a 20-μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), diluted to 100 µl, and stored at -70°C before until use.
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