A Trizol kit (Invitrogen, USA) and Evo M-MLV RT Kit II (Accurate Biotechnology, AG11711, China) were used to extract total RNAs of ‘leaf’ and ‘pet’ samples, which were reverse-transcribed into cDNA. Oligo dT and random 6-mer primers were used for reverse transcription of the lncRNAs and mRNAs. Reverse transcription of the circRNAs was performed using downstream primers in qRT-PCR and random 6-mer primers. The reverse transcription of miRNA was performed using primers downstream of U6 and specific stem-loop primers. A SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) was used to perform qRT-PCR using an ABI 7300 RT-PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The protocols and conditions for qRT-PCR have been described previously, by Shi et al. [66 (link)]. The miRNA expression levels were normalized to those of U6 miRNA, which was used as the endogenous control. The expression levels of the mRNAs, lncRNAs, and miRNAs were normalized to those of actin. The primer sequences used for RT-PCR are listed in Table S8 . qRT-PCRs were performed with three technical replicates and four biological replicates.
Abi 7300 rt pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 7300 RT-PCR system is a real-time PCR instrument designed for quantitative gene expression analysis. It features a 96-well block format and uses fluorescence detection to monitor the amplification of target DNA sequences during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sybr green 1 master mix, by Takara Bio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Evo m mlv rt kit 2, by Accurate Biology (1 mentions)
Sybr green premix pro taq hs qpcr kit, by Accurate Biology (1 mentions)
Sybr exscript rt pcr kit, by Takara Bio (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit with dna eraser, by Takara Bio (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Primescript kit, by Takara Bio (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Prism 7500, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Ex taq, by Takara Bio (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Toyobo (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Omniscript reverse transcription kit, by Qiagen (1 mentions)
13 protocols using abi 7300 rt pcr system
1
Quantitative Transcriptome Analysis of Plant Samples
2
Colon Tissue Analysis of c-kit and PAR-2 Expressions
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Total RNAs were extracted from the colon tissue samples of the rats using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), and RT-qPCR has been performed to examine the expressions of c-kit and PAR-2 in different tissue samples using the SYBR ExScript RT-PCR kit (TaKaRa Bio Inc., Kusatsu, Japan). ABI 7300 RT PCR System (Thermo Fisher Scientific, Waltham, MA, USA) has been used for the amplification process. The thermo-cycling profiles were as follow: 95°C for 30 seconds; followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Primers were all synthesized by Sangon Biotech Engineering (Shanghai) Co. Ltd. (Shanghai, China). The relative expression of c-kit and PAR-2 in each sample was normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the 2−ΔΔCt method. In Figure 1 , the relative expressions of c-kit and PAR-2 in other groups were presented as fold changes of the 2−ΔΔCt value compared to the average 2−ΔΔCt value of the control group, and the average 2−ΔΔCt value of the control group was set as onefold. The sequences of the primers were as follow: PAR-2, forward: 5′-GACTTTCTCTCGGTGCGTCC-3′; reverse: 5′-CCCCATAAATCCAGTTGTTGCC-3′. C-kit, forward: 5′-CCGACGCAACTTCCTTATGAT-3′; reverse: 5′-TCAGGACCTTCAGTTCCGACA-3′, GAPDH, forward: 5′-AGAAGGCTGGGGCTCATTTG-3′; reverse: 5′-AGGGGCCATCCACAGTCTTC-3′.
3
Quantitative Real-Time qPCR Analysis of Antioxidant and Stress Genes
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Quantitative real-time qPCR was carried out with the help of ABI 7300 RT-PCR system (Applied Biosystems) with PowerUPTM SYBR Green Master Mix. Amplification of antioxidant and stress related genes (APX, SOD, POD, CAT, LEA, and DREB) was achieved as per manufacturer’s protocol. PCR reaction of about of 20 μL consisted 12.5 μL of 2X PowerUPTM SYBR Green Master Mix, 10 ng of cDNA template, 300 nM primer (reverse and forward), and nuclease free water. The following conditions were used for thermocycling: 2 min at 50 °C, 10 min at 95 °C and 40 cycles alternating between 15 s at 95 °C and 1 min at 60 °C to verify primer specificity melting curve analysis (65–95 °C) was routinely performed after 40 cycles. A single amplified product for all genes was observed from the melting curves. For each sample three analytical replicates were used for the calculation of expression levels of the individual genes along with tubulin as an internal control and recorded as Ct at the default threshold (0.2). Ct was transformed to quantities relative to the sample and Ct values of the used target genes were normalized using the Ct values of tubulin, mRNA levels of the genes were normalized with that of the tubulin [49 (link)]. 2−ΔΔCt method [50 (link)] was used for the calculation of relative expression levels of the genes in respect to that of control.
4
Quantitative RT-PCR Analysis of S. agalactiae
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S. agalactiae strains GD201008-001, Δxf, CΔxf were cultured overnight in TSB at 37°C and isolated RNA with an E.Z.N.A.™ Bacterial RNA isolation kit (OMEGA, Beijing, China). The cDNA synthesis was performed using the PrimeScript™ RT reagent kit with DNA eraser (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The mRNA levels were measured using two-step relative qRT-PCR. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was amplified as an internal control. The specific primers used for the various RT-PCR assays are listed in Table 1 . The SYBR Green PCR method was performed using the AceQ™ qPCR SYBR™ Green Master Mix (Vazyme Biotech Co., Ltd.). Reactions were carried out in triplicate and repeated three times. An ABI 7300 RT-PCR system (Applied Biosystems) was used for relative qRT-PCR. Dissociation analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The comparative cycle threshold method (2−ΔΔCTmethod) [35 (link)] was used to analyze the mRNA levels.
5
Quantifying mRNA and miRNA Expression
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The total RNA was extracted from these cells utilizing TRIzol reagent (Invitrogen, MA, USA) and complementary DNA (cDNA) synthesis was accomplished using the Prime Script kit (Takara, Beijing, China). The qRT-PCR was performed using SYBR Green (Takara, Beijing, China) in an ABI-7300 RT-PCR system (Applied Biosystems, CA, USA). The ratio to GAPDH was used as an internal control for mRNA, and miRNA expression was normalized utilizing U6. The sequences of the following primers were made:
KIF2C-F: 5′-CAGTGGAATGGGCAGAAGGA-3′
KIF2C-R: 5′-CGGGCAAATTCCCAGTTTGG-3′
miR-186-3p-F: 5′-GCCCAAAGGTGAATTTTTTGGG-3′
miR-186-3p-R: 5′-CAGTGCGTGTCGTGGAGT-3′
GAPDH-F: 5′-GGACTGACCTGCCGTCTAG-3′
GAPDH-R: 5′- TAGCCCAGGATGCCCTTGAG-3′
U6-F: 5′-CTCGCTTCGGCAGCACA-3′
U6-R: 5′-AACGCTTCACGAATTTGCGT-3′
KIF2C-F: 5′-CAGTGGAATGGGCAGAAGGA-3′
KIF2C-R: 5′-CGGGCAAATTCCCAGTTTGG-3′
miR-186-3p-F: 5′-GCCCAAAGGTGAATTTTTTGGG-3′
miR-186-3p-R: 5′-CAGTGCGTGTCGTGGAGT-3′
GAPDH-F: 5′-GGACTGACCTGCCGTCTAG-3′
GAPDH-R: 5′- TAGCCCAGGATGCCCTTGAG-3′
U6-F: 5′-CTCGCTTCGGCAGCACA-3′
U6-R: 5′-AACGCTTCACGAATTTGCGT-3′
6
Quantifying Ki-67 Expression in Breast Cancer Tissues
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Breast cancer tissues were collected from 62 patients during surgery and analyzed. Cancer tissue (200 µg) was homogenized in 1 ml TRIzol solution and RNA was isolated according to the manufacturer's instructions. The isolated RNA was dissolved in 30 µl of diethylpryocarbonate-treated water and quantified using an ultraviolet spectrophotometer (One Drop, Nanjing, China). RNA was reverse transcribed into cDNA and stored in a freezer at −20°C.
The primer sequences used for Ki-67 amplification were: F: 5′-CGTAGCAGCACAGAAAT-3′ and R: 5′-TGATGGTTGAGGTCGTTCCTTGATG-3′ (9 (link)). U6 was used as an internal control, with the following primers: F: 5′-CTCGCTTCGGCAGCACA-3′ and R: 5′-AACGCTTCACGAATTTGCGT-3′. The PCR reaction conditions used were: denaturation at 95°C for 20 sec, followed by 50 cycles of 60°C for 20 sec and 70°C for 1 sec. RT-PCR data were analyzed on an ABI 7300 RT-PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA) and the relative quantitative analysis of gene expression was performed using the 2−ΔΔCt method (10 (link)).
The primer sequences used for Ki-67 amplification were: F: 5′-CGTAGCAGCACAGAAAT-3′ and R: 5′-TGATGGTTGAGGTCGTTCCTTGATG-3′ (9 (link)). U6 was used as an internal control, with the following primers: F: 5′-CTCGCTTCGGCAGCACA-3′ and R: 5′-AACGCTTCACGAATTTGCGT-3′. The PCR reaction conditions used were: denaturation at 95°C for 20 sec, followed by 50 cycles of 60°C for 20 sec and 70°C for 1 sec. RT-PCR data were analyzed on an ABI 7300 RT-PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA) and the relative quantitative analysis of gene expression was performed using the 2−ΔΔCt method (10 (link)).
7
Quantitative RT-PCR Analysis of Wheat Genes
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Quantitative RT-PCR (qRT-PCR) was used to investigate the relative expression levels of TaAGC1 and the genes encoding ROS-scavenging enzymes (POX2, CAT1 and SOD3) and ROS-producing enzyme NADPH oxidase (NOX), and four defence-associated genes (defensin, nsLTP1, chitinase 2 and PR10) in wheat. qRT-PCR was performed using SYBR Green I Master Mix (TaKaRa) in a volume of 25 μl on an ABI 7300 RT-PCR system (Applied Biosystems). Reactions were set up using the following thermal cycling profile: 95°C for 5min, followed by 41 cycles of 95°C for 15 s and 60°C for 31 s. The products were examined using a melting curve analysis program. Three biological replications were run. The relative expression of the target genes was calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)), where the wheat Actin gene, TaActin, was used as the internal reference. RT-PCR was used to investigate the transcription of BSMV CP gene in VIGS experimental wheat plants.
The accession number of all the genes and sequences of all the primers in the study are listed inSupplementary Table S2 .
The accession number of all the genes and sequences of all the primers in the study are listed in
8
Wheat Defense and Monolignol Genes Expression
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Q-RT-PCR analysis with TaCAD12 specific primers TaCAD12-Q-265F265F and TaCAD12-Q-557R was used to investigate the relative transcriptional levels of TaCAD12 in various wheat plants. The tested wheat defense-marker genes include Defensin (NCBI accession no. CA630387), PR10 (NCBI accession no. CA613496), PR17c (NCBI accession no. TA65181), and chitinase1 (Chit1, NCBI accession no. CA665185). The tested wheat monolignol biosynthesis-related genes include TaCAD1 (NCBI accession no. GU563724), TaCCR (NCBI accession no. DQ449508), and TaCOMT1 (NCBI accession no. AY226581). Q-RT-PCR was performed using SYBR Green I Master Mix (TaKaRa) in a volume of 25 μl on an ABI 7300 RT-PCR system (Applied Biosystems). Reactions were set up using the following thermal cycling profile: 95°C for 15 min, followed by 41 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 32 s. The relative transcriptional levels of the target genes was calculated using the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)), where the wheat Actin gene TaActin was used as the internal reference. The relative transcriptional levels of the tested genes in the TaCAD12-overexpressing wheat lines or in BSMV:TaCAD12-infected wheat plants were relative to those in WT recipient or in BSMV:GFP-infected wheat plants.
9
Quantifying miRNA Expression via RT-qPCR
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Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 µg total RNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) and reverse transcribed using the following temperature protocol: 37C for 15 min, 85C for 5 sec and stored at 4C. RT-qPCR was performed using 2X SYBR Green qPCR ProMix (EnzyValley) in an ABI 7300 RT PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction mixtures were incubated in a 96-well plate and the RT-qPCR thermal cycling conditions were as follows: Initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 5 sec and final extension at 40 cycles of 60°C for 30 sec. RNU6B was used as the reference control. Primer sequences are presented in Table I . The relative expression level of miR-499 was determined using the 2−ΔΔCq method (22 (link)).
10
Quantitative Gene Expression Analysis
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cDNA was synthesized using 100 ng total RNA
by SuperScript™ III Reverse Transcriptase (Life
Technologies, USA) and amplified by ExTaq (Takara,
Japan). RT-PCR was performed using Platinum SYBR
Green qPCR SuperMix-UDG plus ROX (Invitrogen,
USA), according to the manufacturer’s instructions in an
ABI7300 RT-PCR System (Applied Biosystems, USA).
Primer sequences were as follows:
GAPDH-
F: 5´-CTCATTTCCTGGTATGACAACGA-3´
R: 5´-CTTCCTCTTGTGCTCTTGCT-3´
FoxP3-
F: 5´-CCAGCCATGATCAGCCTCAC-3´
R: 5´-CCGAAAGGGTGCTGTCCTTC-3´
INF-γ-
F: 5´-GGTTCTCTTGGCTGTTACTG-3´
R: 5´-TCTTTTGGATGCTCTGGTCA-3´
IL-17-
F: 5´-AACCGATCCACCTCACCTTG-3´
R: 5´-CCCACGGACACCAGTATCTT-3´
Real-time PCR data were analyzed by an ABI PRISM 7500 RT-PCR program. RT-PCR program
included polymerase activation and initial denaturation (95˚C, 10 minutes), denaturation
(95˚C, 10 minutes), annealing and extension (60˚C, 60 seconds) for 40 cycles. All of the
absolute data were normalized against a housekeeping gene (GAPDH) and
control group, including embryonic stem cells (ESCs) and mouse embryonic fibroblast (MEF)
using the ∆∆Ct method. The assay was run in triplicate to obtain gene expression data.
by SuperScript™ III Reverse Transcriptase (Life
Technologies, USA) and amplified by ExTaq (Takara,
Japan). RT-PCR was performed using Platinum SYBR
Green qPCR SuperMix-UDG plus ROX (Invitrogen,
USA), according to the manufacturer’s instructions in an
ABI7300 RT-PCR System (Applied Biosystems, USA).
Primer sequences were as follows:
GAPDH-
F: 5´-CTCATTTCCTGGTATGACAACGA-3´
R: 5´-CTTCCTCTTGTGCTCTTGCT-3´
FoxP3-
F: 5´-CCAGCCATGATCAGCCTCAC-3´
R: 5´-CCGAAAGGGTGCTGTCCTTC-3´
INF-γ-
F: 5´-GGTTCTCTTGGCTGTTACTG-3´
R: 5´-TCTTTTGGATGCTCTGGTCA-3´
IL-17-
F: 5´-AACCGATCCACCTCACCTTG-3´
R: 5´-CCCACGGACACCAGTATCTT-3´
Real-time PCR data were analyzed by an ABI PRISM 7500 RT-PCR program. RT-PCR program
included polymerase activation and initial denaturation (95˚C, 10 minutes), denaturation
(95˚C, 10 minutes), annealing and extension (60˚C, 60 seconds) for 40 cycles. All of the
absolute data were normalized against a housekeeping gene (GAPDH) and
control group, including embryonic stem cells (ESCs) and mouse embryonic fibroblast (MEF)
using the ∆∆Ct method. The assay was run in triplicate to obtain gene expression data.
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