The samples collected at 6:00, 12:00, 18:00, and 0:00 h during the full blossoming period were subjected to qRT-PCR tests to determine the transcript abundance of the genes involved in formation of terpenoids. The experiments were performed on Applied Biosystems 7500 Fast Real-Time PCR platform using the SYBR® Premix Ex TaqTM II mix (Takara Biotechnology Co., Ltd., Dalian, Japan) and the results were analyzed by the Applied Biosystems 7500 software (Applied Biosystems Life Technologies). Three biological replicates were tested and relative transcript levels were calculated by the 2-ΔΔCT method using β-Actin as the endogenous control gene for data normalization. The relative gene expression was determined as previously described by Livak and Schmittgen (2001) (link). The primers for qRT-PCR analysis are listed in Supplementary Table S1 .
Sybr premix ex taqtm 2 mix
Manufactured by Takara Bio
Sourced in Japan, United States
SYBR® Premix Ex TaqTM II mix is a ready-to-use qPCR reagent containing SYBR® Green I dye and Taq DNA polymerase. It is designed for sensitive and specific quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Quantstudiotm6 flex real time pcr instrument, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Applied biosystems 7500, by Thermo Fisher Scientific (2 mentions)
Synthesis kit, by Thermo Fisher Scientific (2 mentions)
7500 fast real time pcr platform, by Takara Bio (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by CoWin Biotech (1 mentions)
Rq1 dnase 1, by Promega (1 mentions)
Revertaidtm first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using sybr premix ex taqtm 2 mix
1
Temporal Dynamics of Terpenoid Biosynthesis
2
Quantitative RT-PCR analysis of gene expression
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Total RNA was extracted from peripheral blood, fresh‐frozen biopsies and pWSLV‐02‐LILRA3 plasmid‐ or pWSLV‐02‐transfected U937 cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The quantity and quality of the RNA samples were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Total RNA (1 μg) was used to synthesize cDNA using a first‐strand cDNA synthesis kit (Thermo Scientific). qRT–PCR was subsequently performed using the QuantStudioTM6 Flex Real‐Time PCR instrument (ABI) with the SYBR® Premix Ex TaqTM II mix (Takara, Kusatsu, Japan). The primer sequences are listed in Supporting information, Table S2 . The 2−ΔΔCT method was applied to determine the relative mRNA levels normalized to the housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH).
3
Quantitative Analysis of Gene Expression
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Total RNA was extracted from 30 paired tissues with TRIzol reagent (Invitrogen, USA). Total RNA (1 μg) was used to synthesize first-strand cDNA using a synthesis kit (Thermo Fisher Scientific, USA). The experiments were performed by using a QuantStudioTM 6 Flex Real-Time PCR instrument (ABI, USA) with SYBR® Premix Ex TaqTM II Mix (Takara, Japan). GAPDH was taken as the internal control. The relative mRNA expression levels were calculated using the 2-ΔΔCt method 32 (link). The gene specific primers were as follows: MYL9 (Forward 5'-GGATGTGATTCGCAACGCCTTTG-3' and Reverse 5'-CGGTACATCTCGTCCACTTCCT-3'); CNN1 (Forward 5'-CCAACGACCTGTTTGAGAACACC-3' and Reverse 5'-ATTTCCGCTCCTGCTTCTCTGC-3'); GAPDH (Forward 5'-AGAAGGCTGGGGCTCATTTG-3' and Reverse 5'-GCAGGAGGCATTGCTGATGAT-3').
4
Transcriptome Analysis of Flower Development
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Total RNA was isolated from 0.20 g frozen flowers using TRIzol reagent (CoWin Biotech Co., Ltd., Beijing, China), following the manufacturer’s instructions, and then treated with RQ1 DNase I (Promega, Madison, WI, USA) to remove genomic DNA. To synthesize first-strand cDNA, 3.50 μg total RNA was used with the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific Inc., USA) according to the manufacturer’s instructions. The synthetic first-strand cDNAs were diluted 10-fold for gene expression analysis.
Gene expression was detected by qRT-PCR in both ‘Gecheng’ and ‘Liuye’ flowers at four flowering stages. The qRT-PCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR platform with the SYBR® Premix Ex TaqTM II mix (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer’s instructions, and the results were analyzed using the Applied Biosystems 7500 software (Applied Biosystems Life Technologies). Three biological replicates were tested, and reactions carried out in triplicate. Relative transcript levels were calculated by the 2-ΔΔCt method using β-actin as the endogenous control gene for data normalization. The primers for qRT-PCR analysis are listed in Supplemental TableS2 .
Gene expression was detected by qRT-PCR in both ‘Gecheng’ and ‘Liuye’ flowers at four flowering stages. The qRT-PCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR platform with the SYBR® Premix Ex TaqTM II mix (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer’s instructions, and the results were analyzed using the Applied Biosystems 7500 software (Applied Biosystems Life Technologies). Three biological replicates were tested, and reactions carried out in triplicate. Relative transcript levels were calculated by the 2-ΔΔCt method using β-actin as the endogenous control gene for data normalization. The primers for qRT-PCR analysis are listed in Supplemental Table
5
Quantitative Real-Time PCR Analysis Protocol
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Quantitative real-time PCR analysis (qRT-PCR) analyses were performed using SYBR Premix ExTaqTM II Mix (TaKaRa, Shiga, Japan). GAPDH (glyceraldehyde-3-phosphate dehydrogenase, accession no. DQ355800) was used as a reference. The expression levels of all candidate genes were analyzed by the 2−ΔΔCT CT method. The primers used for qRT-PCR were listed in Supplementary Table S1 [1 (link),2 (link),22 (link)].
6
Quantitative Gene Expression Analysis of Blood and Colon Samples
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Total RNA was extracted from human blood and human or mouse colon samples using Trizol reagent (Invitrogen, USA) according to the manufacturer's protocols. The RNA samples were quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Total RNA (1 μg) was used to synthesise firststrand cDNA using a synthesis kit (Thermo Scientific, USA). qRT-PCR was subsequently performed using a QuantStudio TM 6 Flex Real-Time PCR instrument (ABI, USA) with SYBR ® Premix Ex Taq TM II mix (Takara, Japan). The gene-specific primer pairs that were used in these experiments are listed in Table 2.
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