Leaves of 3-week-old wild-type plants infiltrated with V. vulnificus 96-11-17M (OD600 = 1 in 10% PBS) were harvested at 0, 12, 24, 48 and 72 h post-infiltration and immediately frozen in liquid nitrogen. Subsequently, the leaf tissues were ground to a fine powder using a mortar and pestle. Total RNA was isolated from 100 mg ground tissue using RNeasy® Plus Mini kit, according to the manufacturer’s protocol (Qiagen, CA, USA). First-strand cDNA was synthesized using 2 μg total RNA, oligo-dT primer, dNTPs and Moloney murine leukaemia virus reverse transcriptase (M-MLV RT; Enzynomics, Daejeon, South Korea). To validate the RNA-Seq data, qRT-PCR was performed using a Chromo4 Real-time PCR System (Bio-RAD, CA, USA). Each reaction mixture contained 2× Brilliant SYBR Green Supermix (Bio-RAD, CA, USA), cDNA template and 0.5 μM gene-specific primers. Sequences were amplified using the following conditions: initial denaturation at 95°C for 10 min, followed by 44 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 42 sec. Gene expression levels were normalized relative to the expression of AtACT2 (GenBank accession no. Q96292). Primer sets used in this study are listed in S1 Table .
M mlv rt
Manufactured by Enzynomics
M-MLV RT is a reverse transcriptase enzyme derived from the Moloney Murine Leukemia Virus. It is used to synthesize complementary DNA (cDNA) from RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Chromo4 real time pcr system, by Bio-Rad (3 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Iqtm sybr green supermix, by Bio-Rad (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Topreal qpcr 2x premix, by Enzynomics (1 mentions)
Eco real time pcr system, by Illumina (1 mentions)
Random hexamer, by Takara Bio (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Taq polymerase, by Takara Bio (1 mentions)
7 protocols using m mlv rt
1
Transcriptional Response of Arabidopsis to Vibrio Infection
2
Validating RNA-seq Data by qRT-PCR
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To validate RNA‐seq data, expression of selected DEGs (including defence marker genes CRK, WRKY, and GST) was analyzed by qRT‐PCR. To perform qRT‐PCR, first‐strand cDNA was synthesized from 2 mg of DNase‐treated total RNA using oligo‐dT primers and Moloney murine leukaemia virus reverse transcriptase (MMLV‐RT, Enzynomics, Daejeon, South Korea). PCR reactions were performed according to the manufacturer’s instructions. Expression of DEGs was analyzed using the primers listed in Table S1 . A Chromo4 Real‐Time PCR system (Bio‐Rad, CA, USA) was used for qRT‐PCR. Reaction mixtures contained cDNA template, iQTM SYBR® Green Supermix (Bio‐Rad, CA, USA) and 10 pM of each primer. Thermocycler parameters used for qRT‐PCR were as follows: initial polymerase activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 60 sec, and extension at 72°C for 30 sec. Conditions were determined by comparing threshold values in a series of dilutions of the reverse‐transcribed product, a non‐reverse‐transcribed template control, and a non‐template control for each primer pair. Relative RNA levels were calibrated and normalized relative to the level of AtActin2 mRNA.
3
Pathogen-Induced Gene Expression Analysis
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Following inoculation with pathogen, leaf tissue was harvested 0, 12, 24, 36 and 48 h after inoculation with Pta and used for total RNA isolation. Total RNA was isolated using an RNeasy® Plus Mini kit according to the manufacturer’s protocol (Qiagen, USA). First-strand cDNA was synthesised using 2 μg of RNA, oligo dT primer, dNTP and Moloney murine leukaemia virus reverse transcriptase (M-MLV RT; Enzynomics, Korea). Quantitative reverse-transcription PCR (qRT-PCR) was carried out using a Chromo4 real-time PCR system (Bio-Rad, USA). The reaction mixture contained 2× Brilliant SYBR Green qRT-PCR Supermix (Bio-Rad), cDNA and 0.5 μM of each gene-specific primer. The expression of candidate priming genes was analysed using the following primers: 5′-AATATCCCACTCTTGCCG-3′ (NbPR1a-F), 5′-CCTGGAGGATC ATAGTTG-3′ (NbPR1a-R), 5′-ACCATCAGACCAAGATGT-3′ (NbPR2-F) and 5′-TGGCTAAGAGTGGAAGGT-3′ (NbPR2-R) (Kim et al., 2003 (link)). RNA levels were calibrated and normalised relative to the level of NbACT mRNA (GenBank accession no. U60489). The sequences were amplified using the following thermocycler parameters: 10 min at 95°C, followed by 44 cycles of 30 s at 95°C, 30 s at 60°C and 42 s at 72°C.
4
Quantitative RT-PCR Analysis of APIP and ADORA2B
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Total RNA was isolated with TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was performed with Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Enzynomics). Transcript levels were determined by real-time PCR using a PowerUpTM SYBRTM Green Master Mix (Thermo Fisher Scientific) using the following synthetic oligonucleotides sets: human APIP #1 5′-GCGCAGGACAAGGAG CAT-3′ (forward), 5′-TTCTTCGAT GGCGAAGGTCC-3′ (reverse), human APIP #2 5′-GCGCAGGACAAGGAGCA-3′ (forward), 5′-CGATGGCGAAGGTCCACTTA-3′ (reverse); mouse Apip #1 5′-GCTCTCTCGCCTAATAGCGT-3′ (forward), 5′-TCTGGCTGAATGCGTTCCTT-3′ (reverse), mouse Apip #2 5′-TCTGGCTGTCAAGCTCAAGG-3′ (forward), 5′-CGCCTGAGGGAGCAATGTAG-3′ (reverse); rat Apip 5′-TAAGTGGGCCTCCAGCATCTA-3′ (forward), 5′-TCCTGTCCTGGAAACAGAAGG-3′ (reverse), rat Gapdh 5′-CAACTCCCTCAAGATTGTCAGCAA-3′ (forward), 5′-GGCATGGACTGTGGTCATGA-3′ (reverse); human ADORA2B 5′-ATGGAACCACGAATGAAAGC-3′ (forward), 5′-ATGTAGCTCATGGGGACCAC-3′ (reverse); mouse Adora2b 5′-TGCATGCCATCAACTGTATC-3′ (forward), 5′-TGGAAACTGTAGCGGAAGTC-3′ (reverse). Normalization of the gene of interest in samples was calculated using the ΔCt values as previously described23 (link).
5
RT-PCR Analysis of BNIP3 Expression
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Total RNA was obtained using the RNeasy mini kit according to the manufacturer’s procedure (Qiagen, Valencia, CA, United States). Total RNA was converted to cDNA using M-MLV RT (Enzynomics, Daejeon, South Korea), RNase inhibitor and random hexamers (TaKaRa, Otsu, Japan). Negative control reactions were performed as described above, with omission of the enzyme or cDNA. Standard cycling was performed with 35 cycles of 95, 55, and 72°C steps of 30 s each. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel in Tris acetate-EDTA buffer [TAE; Tris base, glacial acetic acid, 0.5 M EDTA (pH 8.0)]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were used as internal controls. The primer sequences used for RT-PCR were as follows: mouse BNIP3: forward, 5′-AGAACCTGCAGGGCTCCTGG-3′ and reverse, 5′-GAAGTTGTCAGACGCCTTCC-3′; mouse GAPDH: forward, 5′-TGCTGATGTCGTGGAGTCT-3′ and reverse, 5′-AATGGGAGTTGCTGTTGAAGTC-3′.
6
Gene Expression Quantification by Real-Time PCR
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The total RNA was synthesized into first-strand complementary DNA (cDNA) using a random primer (9-mer, Invitrogen, Carlsbad, CA, USA) and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Enzynomics Co., Daejeon, Republic of Korea) (Abdellaoui et al. 2023 (link)). The gene expression was quantified using TOPreal™ qPCR 2X PreMIX (Enzynomics Co.), the Eco Real-Time PCR system (Illumina, San Diego, CA, USA), and the following specific primers: mouse Interleukin (IL)-1β (Gene Bank ID, NM_008361, 133 bp), 5′-CCC AAG CAA TAC CCA AAG AA-3′ and 5′-GCT TGT GCT CTG CTT GTG AG-3′; mouse IL-6 (NM_031168, 102 bp), 5′-CCG GAG AGG AGA CTT CAC AG-3′ and 5′-TCC ACG ATT TCC CAG AGA AC-3′; mouse IL-10 (NM_010548, 142 bp), 5′-GCC TGG CTC AGC ACT GCT AT-3′ and 5′-GAA GGC AGT CCG CAG CTC TA-3′; mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh, NM_001289726, 223 bp), 5′-AAC TTT GGC ATT GTG GAA GG-3′ and 5′-ACA CAT TGG GGG TAG GAA CA-3′; human IL-1β (NM_000576, 180 bp), 5′-CTG TCC TGC GTG TTG AAA GA-3′ and 5′-TTC TGC TTG AGA GGT GCT GA-3′; human IL-6 (NM_000600, 175 bp), 5′-TAC CCC CAG GAG AAG ATT CC-3′ and 5′-TTT TCT GCC AGT GCC TCT TT-3′; human IL-10 (NM_000572, 253 bp), 5′-TGA GAA CAG CTG CAC CCA CT-3′ and 5′-GTT CAC ATG CGC CTT GAT GT-3′; human Gapdh (NM_001256799, 278 bp), 5′-ACT GGC GTC TTC ACC ACC AT-3′ and 5′-GGG CCA TCC ACA GTC TTC TG-3′.
7
RNA Extraction and RT-PCR Analysis
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The RNA extraction was performed using an RNeasy mini kit (Qiagen, USA) according to the manufacturer’s instruction. Briefly, 2 μg of total RNA was reverse transcribed using M-MLV RT (Enzynomics, Korea) in a reaction including RNase inhibitors and a random hexamer (Takara, Japan) according to the manufacturer’s instructions. The PCR reaction was performed with forward and reverse primers and 0.5 μl of Taq polymerase (Takara, Japan). The nucleotide sequences of the RT-PCR primers and the sizes of amplified products are described in Supplementary Table S1 . The amplified products were separated on 1.5% agarose gel and visualized after staining with ethidium bromide (Sigma-Aldrich, USA). The expression of the housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin was determined as an internal control.
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