Total RNA was extracted from grapevine using the CTAB method with minor modification. The cDNA synthesis was conducted according to PrimeScript™ RT reagent Kit (Takara, Kusatsu, Japan). Three biological cDNA replicates were mixed for next amplification. Specific primers listed in Supplementary Materials Table S1 were designed for real-time PCR. The amplification was performed on the QIAGEN Rotor-Gene Q system (QIAGEN, Hilden, Germany) using the SuperReal PreMix Plus (SYBR Green) kit (TIANGEN, Beijing, China). Constitutively expressed elongation factor1-α (EF1-α) was used as reference gene to calculate relative expression levels with the 2−∆∆Ct method. All reactions were performed with three technical replicates.
Rotor gene q system
Manufactured by Qiagen
Sourced in Germany, United States, France, China, United Kingdom, Japan, Denmark
The Rotor-Gene Q system is a real-time PCR cycler designed for high-performance nucleic acid quantification and analysis. It features a unique rotary design and advanced optical system to deliver reliable and consistent results across a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (22 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (18 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (14 mentions)
Rneasy mini kit, by Qiagen (11 mentions)
Trizol, by Thermo Fisher Scientific (10 mentions)
Rotor gene sybr green pcr kit, by Qiagen (9 mentions)
Rotor gene sybr green pcr master mix, by Qiagen (8 mentions)
Kapa sybr fast qpcr kit, by Roche (7 mentions)
Quantitect reverse transcription kit, by Qiagen (6 mentions)
Random hexamer, by Roche (6 mentions)
Rt2 first strand kit, by Qiagen (5 mentions)
Qiaamp dna mini kit, by Qiagen (5 mentions)
Miscript sybr green pcr kit, by Qiagen (5 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Rotor gene 1, by Qiagen (4 mentions)
Lunascript rt supermix, by New England Biolabs (4 mentions)
Luna universal qpcr master mix, by New England Biolabs (4 mentions)
Gene specific primer pairs, by Eurofins (4 mentions)
193 protocols using rotor gene q system
1
Grapevine Total RNA Extraction and qRT-PCR
2
Profiling PvNLPs in Grapevine Under P. viticola Infection
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To monitor PvNLPs transcript profiling during P. viticola infection of grapevine, leaf inoculation using spore drops of P. viticola ZJ-1-1 and total RNA was extracted using the CTAB procedure were conducted as previous described.35 (link) For RNA isolation of N. benthamiana, a commercial kit (RNA simple Total RNA Kit, Tiangen) was used following the recommended protocols. All cDNA synthesis and quantitative RT-PCR reaction were performed by using protocols established in our lab. Briefly, the total RNA was used as a template for reverse transcription with a RevertAid TM First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). The qRT-PCR was performed on the QIAGEN Rotor-Gene Q system (QIAGEN, Hilden, Germany) using a SuperReal PreMix Plus (SYBR Green) kit (TIANGEN Biotech Co., Ltd., Beijing, China). Primers (Table S1) were designed to anneal specifically to each targeted gene and by using Beacon Designer 8.14 software with default setting for SYBR Green real-time PCR. The PvActin gene36 (link) and NbEF1α gene35 (link) were used as constitutively expressed endogenous controls to determine relative expression values by the 2−ΔΔCt method37 (link) for P. viticola and N. benthamiana, respectively.
3
Quantifying RPW8 Transcription in Grapevine-Downy Mildew Interactions
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To determine the transcription levels of the five RPW8s during the infections of V. pseudoreticulata and V. vinifera with P. viticola, leaves were excised and used for RNA extraction. The qRT-PCR was performed on the QIAGEN Rotor-Gene Q system (QIAGEN, Hilden, Germany) using a SuperReal PreMix Plus (SYBR Green) kit (TIANGEN Biotech Co.， Ltd., Beijing, China). To calculate the transcription levels, constitutively expressed elongation factor1-α (EF1-α) was used as an internal gene for normalization [37 (link)] with the 2−∆∆Ct method [38 (link)]. All reactions were performed in triplicate.
4
Quantitative Analysis of Matrisomal Genes
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Quantitative real-time PCR was performed for common matrisomal genes, where GAPDH was used as an internal control for the normalization of RT qPCR data. Total RNA was isolated using RNAiso Plus from MeT-5A monolayers, after which 1 μg of total RNA was reverse transcribed to cDNA using Verso cDNA Synthesis kit as per the manufacturer’s protocol (AB-1453; Thermo Fisher Scientific). Real-time qPCR was performed on QIAGEN Rotor-Gene Q System (QIAGEN) using a standard two-step amplification protocol followed by a melting curve analysis. The amplification reaction mixture (total volume of 10 μL) contained 10 ng of cDNA, 5 μL 2 × DyNAmo Flash SYBER Green master mix, and 0.25 μM of the appropriate forward and reverse primer. Primer sequences of GAPDH and matrisomal genes are mentioned in the Table S1 . Relative gene expression was calculated using the comparative Ct method, and gene expression was normalized to non-senescent MeT-5A cells. Appropriate no template and no RT controls were included in each experiment. All the samples were analyzed in triplicates and repeated at least three times independently.
5
Quantitative SARS-CoV-2 Detection Protocol
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The PCR master mixture was prepared using a SentiFAST Probe No‐ROX One Step Kit (Bio Line, USA) and primer‐probe mix targeting N1 and N2 gene regions selected from WHO and USA‐CDC sources. The PCR was performed on Rotor‐Gene Q System (Qiagene, USA) where the PCR program was as follows: Initial step, one cycle at 45°C for 600 seconds, one cycle at 95°C for 120 seconds, 45 cycles at 95°C for 10 seconds, and 55°C for 30 seconds. TaqMan probes are labeled at the 5′‐end with the reporter molecule 6‐carboxyfluorescein (FAM). Copies/μL values of the standards prepared by serial dilution of the synthetic gene with a certain number of copies (200 000 /μL) are shown in Figure 1 .
The synthetic gene (GenBank: NC_045512.2) copy/μL value was calculated with the formula [(amount × 6.022 × 1023)/(length × 1 × 109 × 650)]. Samples with known copies/μL values obtained by serial dilution from the synthetic gene of 200 000 copies/μL were included in the study. In the PCR study, standards were run in sample conditions and the values were recorded in the software of the device. The formulation created by the device according to the recorded standard values was applied on the samples and the copy/μL values were obtained.
The synthetic gene (GenBank: NC_045512.2) copy/μL value was calculated with the formula [(amount × 6.022 × 1023)/(length × 1 × 109 × 650)]. Samples with known copies/μL values obtained by serial dilution from the synthetic gene of 200 000 copies/μL were included in the study. In the PCR study, standards were run in sample conditions and the values were recorded in the software of the device. The formulation created by the device according to the recorded standard values was applied on the samples and the copy/μL values were obtained.
6
Carob Moth Heat Stress Response
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RNA and cDNA was prepared from pools of 10 larvae for each stress and control treatment. RT-qPCR was employed to determine the expression of HSP70 and HSP90 in treated and non-treated carob moth using RealQ Plus 2x Master Mix Green (ampliqon) on a Rotor-Gene® Q system (QIA-Gene). Gene-specific primers (Table 1 ) were designed using Primer-Blast. A ribosomal protein gene, RL32, which is constitutively expressed in all tissues, was used as an internal control. The PCR amplifications were performed with the following cycling conditions: one cycle at 95°C (15 min), followed by 40 cycles of denaturation at 95°C (15 s), annealing at 55°C for the 20s, and extension at 72°C for 20 s. Quantitative analysis followed a comparative CT (ΔΔCT) method [39 (link)]. A melt curve analysis was used to assess whether a single amplicon was produced in all RT-qPCR reactions. For each treatment, three replicates of 10 individuals were analyzed.
7
CXCR4 Expression Analysis by qRT-PCR
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Paired oligonucleotide forward and reverse primers (Table S2 ) for C-X-C chemokine receptor type 4 (CXCR4) and GAPDH were designed using Primer Designer (Scientific and Educational Software, Durham, USA) against the sequence downloaded from GenBank and obtained from Invitrogen. The process of RNA extraction and cDNA generation was the same as those in the PCR array experiment. The cDNA sample was mixed with forward and reverse primers and SYBR Green ROX qPCR Mastermix (QIAGEN, West Sussex, UK). The PCR mixture was processed and analyzed with the Rotor-Gene Q system (QIAGEN, West Sussex, UK). All mRNA data were expressed relative to the endogenous control gene, GAPDH.
8
Rapid Detection of EVE and EVEX
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For the detection of EVE and EVEX, we used Taqman real-time PCR (rPCR) on the Rotor-Gene Q system (Qiagen, Hilden, Germany) using the QIAGEN OneStep RT-PCR kit according to the manufacturer’s instructions. For EVE we used primers and probes developed by Janssen [35 ], and for EVEX, primers and probes developed by [27 (link)].
9
RT-qPCR for Gene Expression Analysis
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The cDNAs used for RT-qPCR were synthesized using the SuPrimeScript RT Premix Kit and oligo dT primer (GeNet Bio, South Korea). RT-qPCR was performed using the real-time cycler (Rotor-Gene Q System, Qiagen) at 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s. Then, 2X Prime Q-Mastermix (GeNet Bio, South Korea) containing SYBR Green1 was used for the RT-qPCR. A rice ubiquitin gene (OsUbi1, LOC_Os03g13170) was used as an internal control, and the relative expression values were calculated by the 2−ΔΔCt method. Primer information is presented in Supplementary Table S5 .
10
Quantifying Gene Expression with qRT-PCR
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Gene expression was quantified using the Rotor-Gene Q system (Qiagen) in the presence of SYBR green (Qiagen). cDNA was mixed with master mix, forward and reverse primers each and of probe. Paired oligonucleotide primers were designed for vascular endothelial growth factor A (VEGF-A), matrix metalloproteinase 11 (MMP-11), transforming growth factor beta-1 (TGFβ1), chemokine (C-X-C motif) receptor 2 (CXCR2) and β-actin using Primer Designer (Scientific and Educational Software, Durham, USA) against the sequence downloaded from GenBank and were supplied by Invitrogen. The primer sequences, r2 values and efficiencies are summarised in Table 3 . All TaqMan probes were supplied by Qiagen. All mRNA data were expressed relative to the endogenous control gene β-actin.
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