Total RNA was extracted from three tissue replicates using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). Primers were designed using Primer Premier 5.0, and the sequences are listed in Supplemental Table 22 . qRT-PCR was performed using the ABI 7300 Real-time PCR System (Framingham, MA) with SYBR Green PCR Master Mix (TaKaRa), following procedures described previously (Wang et al., 2015 (link)). All PCR assays were performed in triplicate. The reference gene was 18S rRNA. Relative expression levels were quantified with the 2−ΔΔCt method (Wang et al., 2014 (link)).
Abi 7300 real time pcr system
Manufactured by Takara Bio
Sourced in Japan
The ABI 7300 Real-time PCR System is a laboratory instrument designed for real-time quantitative PCR (qPCR) analysis. It is capable of detecting and quantifying target DNA sequences in real-time during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq, by Takara Bio (3 mentions)
Sybr green pcr master mix, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Primescript 2 first strand cdna synthesis kit, by Takara Bio (1 mentions)
Transzol up plus rna kit, by Transgene (1 mentions)
Amfirivert cdna synthesis platinum master mix, by GenDEPOT (1 mentions)
Tb green premix ex taqtm, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Trizol rna extraction reagent, by Takara Bio (1 mentions)
Virus nucleic acid extraction kit 2, by Geneaid (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Eastep super total rna extraction kit, by Promega (1 mentions)
Drr037a, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
9 protocols using abi 7300 real time pcr system
1
Quantifying Plant Gene Expression
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was prepared at 4 weeks per group after treatment and quantitative reverse transcription (qRT)-PCR was carried out as previously described.21 (link) Before RNA extraction, samples (n=3 for each group) were immediately crushed into powder in a mortar containing liquid nitrogen using the pestle and then mixed with the monophasic solution of phenol and guanidine thiocyanate. Total RNA was extracted using Trizol (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Then, the PrimeScript II First Strand cDNA Synthesis Kit was used to synthesize cDNA from RNA (Takara Bio, Japan). Real-time fluorescence quantitative PCR was performed on an ABI 7300 Real-Time PCR system using the SYBR Premix Ex Taq (Takara Bio, Shiga, Japan). The primers were synthesized by Genecreate Biotechnology Inc. (Wuhan, People’s Republic of China). All mRNA levels were normalized by the housekeeping gene β-actin. The relative quantity of mRNA was calculated using the 2−∆∆Ct method. The primers used for qRT-PCR are listed in Table 1 .
3
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from the three replicates using the TransZol Up Plus RNA Kit (Transgen Biotech, Beijing). Primers were designed using Primer Premier 5.0, and the sequences are listed in Table S27 . qRT-PCR was performed using the ABI 7300 Real-time PCR System (Framingham, MA, USA) with SYBR Green PCR Master Mix (TaKaRa) following procedures described previously80 (link). All PCR assays were performed in triplicate. The reference gene was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative expression level was quantified via the 2−ΔΔCt method81 (link).
4
Quantitative Real-Time PCR for SPARC Expression
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Total RNA was isolated from cells (U87MG, U87MG-shSPARC, PC3, MDA-MB 231, A549) using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was performed with 2 μg of total RNA using amfiRivert Platinum cDNA synthesis Master Mix (GenDEPOT, Barker, TX, USA). From synthesized cDNA, the mRNA expression level of SPARC and GAPDH were detected using TB GreenTM Premix EX TaqTM (#RR420A; TAKARA Bio Inc., Kusatsu, Japan) and analyzed by ABI 7300 Real-Time PCR system. The sequences of the forward and reverse primers of SPARC were 5′-GG TTC AAA CTT TTG GGA GCA-3′ and 5′-CC GAT TCA CCA ACT CCA C-3′. In addition, the sequences of the forward and reverse primers of GAPDH were 5′- TG CAC CAC CAA CTG CTT AGC 3′ and 5′-GG CAT GGA CTG TGG TCA TGA G-3′.
5
Transcriptional response of cotton to Verticillium dahliae
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Cotton RNA was extracted from roots, stems, euphyllas, cotyledons, and stem apices from Xinhai 15 and Xinhai 16 at the two-true-leaf growth stage. Each Xinhai 15 seedling was inoculated with V. dahliae spore suspension (2 × 107 spores/ml) through injured roots, while control seedlings were treated with distilled water in the same way. Euphyllas were collected from three repeats after treatment for 0, 1, 4, 8, 12, 24, 36, 48, and 72 hr. RNA was isolated from leaves and qRT-PCR was performed.
qRT-PCR was performed with three replicates using an ABI 7300 Real Time PCR system and SYBR Premix Ex Taq (Takara). The cotton ubiquitin gene (GbUBQ7F: 5′-GAAGGCATTCCACCTGACCAAC-3′; GbUBQ7R: 5′- CTTGACCTTCTTCTTCTTGTGCTTG-3′) was used as a standard control. Gene-specific primers (qGbNAC1F: 5′-GACCTTGAGCCTTGGGACC-3′; qGbNAC1R: 5′-CTTCCCTCTCTTGTCTTGGTGTA-3′) were used for amplification.
qRT-PCR was performed with three replicates using an ABI 7300 Real Time PCR system and SYBR Premix Ex Taq (Takara). The cotton ubiquitin gene (GbUBQ7F: 5′-GAAGGCATTCCACCTGACCAAC-3′; GbUBQ7R: 5′- CTTGACCTTCTTCTTCTTGTGCTTG-3′) was used as a standard control. Gene-specific primers (qGbNAC1F: 5′-GACCTTGAGCCTTGGGACC-3′; qGbNAC1R: 5′-CTTCCCTCTCTTGTCTTGGTGTA-3′) were used for amplification.
6
Osteogenic Differentiation Gene Expression
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After 7 days' culture, the total RNA was extracted using Trizol RNA extraction reagent (TAKARA, Dalian, Liaoning, China). cDNA was synthesized with PrimeScript RT Master Mix (TAKARA). RT-PCR was performed by ABI 7300 Real-Time PCR System using Premix Ex Tag (TAKARA). The cycling parameters used were as follows: denaturation at 95°C for 15 min and 30 amplification cycles (15 s at 95°C, 25 s at 60°C, and 10 s at 72°C). The primers were RUNX2, COL1A2, osteopontin (OPN), and osteocalcin (OCN) (Table 1 ). Expression of β-actin was used as an internal control.
7
Quantitative RT-PCR for Viral RNA Detection
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The tissues were homogenized in lysates at a ratio of 1:1 (g/ml) and centrifuged at 10,000 g for 30 min, and the supernatants were collected for viral RNA extraction using a Virus Nucleic Acid Extraction Kit II (Geneaid, Taiwan). Quantitative RT-PCR (RT-qPCR) was performed to measure the viral loads in the above-mentioned organs and blood samples. The RNA from the samples was reverse-transcribed and run in an ABI 7300 Real-time PCR System using a SYBR1 Premix Ex Taq™ (Perfect Real Time) kit (TaKaRa, Dalian). The following primers were designed and used on the matrix gene region: 5′-TCTATCGTCCCATCAGGC/GGTCTTGTCTTTAGCCATTC-3′. Simple Vector pMD19-T (50 ng/μl; TaKaRa, Dalian) containing the target virus sequence was used as a reference standard. Series of eight 10-fold dilutions equivalent to 1×103-1×1010 copies per reaction were prepared to generate calibration curves and were run in parallel with the test samples [28 (link)].
8
RNA Extraction and qPCR Analysis
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After bone marrow removal, total RNA was extracted from the distal metaphyses of right femurs using Trizol reagent, following the protocol of the Eastep Super Total RNA Extraction Kit (Promega, Shanghai, China). Then, the PrimeScript RT reagent kit (Takara-Bio, Otsu, Japan) was used to synthesize cDNA from RNA. Polymerase chain reaction tests were conducted using an ABI 7300 Real-Time PCR system using the SYBR Premix Ex Taq II kit (Takara-Bio, Otsu, Japan). The primers were synthesized (Qinke Biotech, Beijing, China) and the sequences are listed in Table 1. Each RNA quantification was carried out in triplicates in a 96-well plate, and the operation was performed on each sample three times. The relative mRNA expression levels were normalised to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample and analysed using the 2-ΔΔCt relative quantification method.
9
Quantification of ALK5, NOX2, and NOX4 mRNA
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Real-time PCR was used to analyze ALK5, NOX2, and NOX4 mRNA levels in brain tissue. Total RNA was extracted by using TRIzol reagent (TakaRa Biotechnology Co., Ltd., Dalian, China) and the concentration and purity of RNA was determined spectrophotometrically. Briefly, 200 ng of RNA from each sample was used for reverse transcription by using a transcription Kit (DRR037A; TaKaRa). Real-time PCR was used to quantitatively determine ALK5, NOX2, and NOX4 mRNA expression levels with SYBR Premix Ex Taq (TaKaRa) using the ABI 7300 Real-Time PCR System. PCR primers for ALK5, NOX2, NOX4, and β-actin are shown in Table 1. Data analysis was performed with the comparative Ct method by using the ABI software. Results were adjusted with the ratio of TGF-β, NOX2, and NOX4 mRNA to β-actin mRNA.
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