Quantitative RT-PCR was employed to quantify the expression levels of genes selected from the microarrays data. RNA from the same 10 d roots samples used for microarray analysis as well as additional experimental replications were subjected to this confirmation. The Applied Biosystems Prism 7700 Sequence Detection System was used for the qRT-PCR analysis (Paul et al., 2004 (link)). The fluorescently tagged probes and paired primers flanking a 60–100 bp section of the gene of interest are listed in Table 2 . The gene expression level was normalized by relating the Taqman results to a standard curve. UBQ11 (AT4G05050) was used as the internal control. Three replicates for each sample were used.
Prism 7700 sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PRISM 7700 Sequence Detection System is a real-time PCR instrument designed for gene expression analysis and genetic variation detection. It utilizes fluorescence detection technology to monitor the amplification of DNA sequences during the PCR process. The system provides accurate quantification of target nucleic acids in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Superase in, by Thermo Fisher Scientific (2 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Molecular Research Center (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Sds software, by Thermo Fisher Scientific (2 mentions)
96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy mini, by Qiagen (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Tri reagent, by Zymo Research (1 mentions)
User bulletin no 2, by PerkinElmer (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Taqman master mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
15 protocols using prism 7700 sequence detection system
1
Quantitative RT-PCR to Validate Microarray Data
2
Quantifying RhCMV DNA in Plasma
3
Quantitative Real-Time PCR Protocol
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cDNA was prepared from total RNA, using a combination of oligo-dT, random primers, dNTPs, and Superscript II (Invitrogen) and Superase.in (RNA inhibitor from Ambion). PCR primers for each gene were obtained from PrimerBank or were designed using Primer3 software, with a melting temperature at 58–60°C and a resulting product of approximately 100 bp. Each PCR was carried out in triplicate in a 20µl volume using SYBR Green Master Mix (Applied Biosystems, CA) for 15 minutes at 95°C for initial denaturing, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s in the ABI prism 7700 sequence Detection system. cDNA prepared from Universal RNA (Stratagene, La Jolla, CA, USA) was used to construct a standard curve for each gene. Two independent experiments were done. Values for each gene were normalized to expression levels of 18S RNA. Primer sequences are available upon request.
4
Quantitative RT-PCR for Mouse Ryk Expression
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Total RNA was extracted using Qiagen RNeasy mini or micro columns. One mirogram of total RNA was used as a template for cDNA synthesis, using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), Oligo dT, and random hexamer primers. The RT-PCR reaction was performed using TaqMan Universal Mastermix (Applied Biosystems, Foster City, CA, USA) and was run on a PRISM 7700 sequence detection system containing a 96-well thermal cycler (Applied Biosystems). The following primers were used in combination with FAM-labeled probes from the universal probe library (Roche, Almere, Netherlands): mouse Ryk forward primer: 5′-CAAGCTTCGAGGTCTGCAC-3′ reverse primer: 5′-ACCATGGGCTTTTCTCCTTC-3′. RQ-PCR results were normalized to Abl expression in the same sample: forward primer: 5′-TGGAGATAACACTCTAAGCATAACTAAAGGT-3′ reverse primer: 5′-GATGTAGTTGCTTGGGACCCA-3′ and probe: 5′-FAM-CCATTTTTGGTTTGGGCTTCACACCATT- NFQ-3′.
5
Quantitative RT-PCR for MDCK Cells
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Total RNA was extracted from MDCK cell lysates with TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed with the PrimeScript cDNA Synthesis kit (Takara Shuzo Co., Otsu, Shiga, Japan). Quantitative real time RT-PCR was performed on the PRISM 7700 Sequence Detection System (Applied Biosystems) using the SYBR green PCR Master Mix (Applied Biosystems) and gene specific primers. The primer sets used in the RT-PCR are summarized in Table 1 . Results were calculated by the comparative Ct method for relative quantification of gene expression and normalization with respect to GAPDH expression.
6
Quantitative Real-Time PCR Protocol
7
Quantitative gene expression analysis
8
Quantifying Gene Expression in Neospora-Infected Mouse Brains
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Steady state mRNA expressions of the genes that upregulated by N. caninum infection and downregulated in symptomatic mice infected with the parasites were measured by qRT-PCR. Total RNA was extracted using TRI Reagent (Sigma, St Louis, MO, USA), from the left halves of brains. Reverse transcription was performed using Superscript III™ Reverse Transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instruction. Real-time PCR was performed using an Applied Biosystems Prism 7700 Sequence Detection System with SYBR Green master mix (AB Applied Biosystems, Life Technologies, Carlsbad, CA, USA). The fold change Ct method was used, where Ct is the threshold concentration (User Bulletin no. 2; Perkin-Elmer, Boston, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control. Specific primer sequences were shown in Table S5 .
9
Quantifying Chlamydia trachomatis Gene Expression
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Endocervical swab samples in MasterPure lysis solution (Epicenter, Illumina) were processed for total nucleic acid according to the manufacturers instructions and stored at −80°C until use. For each patient, 30% of each sample was used to determine C. trachomatis genome copy number. RNA was removed by digestion with RNaseA, the DNA was re-purified by isopropanol precipitation and samples (150 ng/well) were then analyzed by Taqman™ qPCR in triplicate, including a no-template control, using an Applied Biosystems PRISM 7700 Sequence Detection System. The remaining total nucleic acid was treated with DNase and re-precipitated. Each of the samples was subjected to qRT-PCR using Taqman primer/probe sets specific for the mRNAs encoded by the genes euo and omcB. Equivalent amounts of RNA (20 ng) were used to measure expression levels in triplicate and a no-RT control was included for each primer/probe set. Normalization was performed using C. trachomatis genome copy numbers, as previously described (Ibana et al., 2011 (link)).
10
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from cell lysates using the TRIreagent (Molecular Research Center Inc., Cincinnati, OH, USA) according to the provider's instructions. One microgram of total RNA was reverse transcribed using the Prime Script cDNA Synthesis kit (Takara Shuzo Co., Otsu, Shiga, Japan). Quantitative real‐time RT‐PCR was performed using gene‐specific primers and the SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in PRISM 7700 Sequence Detection System (Applied Biosystems) as previously described 12 . All samples were quantified using the comparative Ct method for relative quantification of gene expression. The expression of genes of interest was normalized to that of β‐actin. The primer sets used in this study are listed in Table S1.
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