For qRT-PCR, total mRNA was isolated and cDNA was synthesized accordingly. The specific primers for qRT-PCR are shown in Table S3 . The qRT-PCR condition was: 30 s at 95℃, followed by 40 cycles of 5 s at 95℃, 15 s at 60℃ and 12 s at 72℃, and a final 5 s at 72℃. qRT-PCR was performed using SYBR Premix EX Tag™ (TaKaRa) on a CFX96™ real-time system (Bio-rad). The cycle threshold (CT) values of each sample were standardized using GmActin11 and the relative fold change (FC) of gene expression was calculated based on the 2−ΔΔCT method [52 (link)].
Sybr premix ex tag
Manufactured by Takara Bio
Sourced in Japan, China, United States
SYBR Premix Ex Taq is a ready-to-use real-time PCR master mix that contains SYBR Green I dye, Taq DNA polymerase, dNTPs, and buffer components. It is designed for fast and sensitive real-time PCR amplification and detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Primescript rt reagent kit, by Takara Bio (12 mentions)
Primescript rt kit, by Takara Bio (4 mentions)
Trizol kit, by Thermo Fisher Scientific (3 mentions)
Primer script rt enzyme mix, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Takara Bio (3 mentions)
Rnaiso plus reagent, by Takara Bio (3 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rnaiso reagent, by Takara Bio (2 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Primerscript rt reagent kit, by Takara Bio (2 mentions)
Minibest universal rna extraction kit, by Takara Bio (2 mentions)
7500 system, by Thermo Fisher Scientific (2 mentions)
Cdna synthesis kit, by Toyobo (2 mentions)
Dice tp 800 thermal cyclear, by Takara Bio (2 mentions)
Trizol, by Merck Group (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
53 protocols using sybr premix ex tag
1
Quantitative Real-Time PCR Analysis
2
Quantitative RT-PCR Analysis of Silkworm Genes
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Total mRNA was extracted from midguts with RNeasy Mini Kit (Qiagen, Germany). The RNA sample was digested with RNase-free DNase I at 37°C for 20 min to remove contaminated DNA. Subsequently, RNA was further purified with phenol–chloroform and precipitated with ethanol. The RNA precipitate was dissolved in DEPC-treated ddH
2O and M-MLV RTase (TaKaRa, Japan), and oligo-dT were used to synthesize cDNAs, following the manufacturer's instructions.
qRT-PCR was carried out on Bio-Rad CFX384 real-time system (Bio-Rad) and SYBR Premix Extag (TaKaRa, Japan) with SYBR Green I as a fluorescent dye. The primers were designed with Primer 5.0 software and are listed in
Table 1 . The mRNA of the housekeeping gene,
B. morisilkworm translation initiation factor (
BmTIF) was used as inner standard. The PCR amplification condition was 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 55°C for 30 s, 72°C for 30 s, and finally hold at 4°C. Three parallel experiments were performed. The result was expressed as ratio on
BmTIF(inner standard) mRNA, and the ratio of detected gene over
BmTIFin male silkworm was arbitrarily set as 1 (
Canbay et al. 2003 (link)
). The statistic significance of difference was analyzed with one sample
t-test.
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Total mRNA was extracted from midguts with RNeasy Mini Kit (Qiagen, Germany). The RNA sample was digested with RNase-free DNase I at 37°C for 20 min to remove contaminated DNA. Subsequently, RNA was further purified with phenol–chloroform and precipitated with ethanol. The RNA precipitate was dissolved in DEPC-treated ddH
2O and M-MLV RTase (TaKaRa, Japan), and oligo-dT were used to synthesize cDNAs, following the manufacturer's instructions.
qRT-PCR was carried out on Bio-Rad CFX384 real-time system (Bio-Rad) and SYBR Premix Extag (TaKaRa, Japan) with SYBR Green I as a fluorescent dye. The primers were designed with Primer 5.0 software and are listed in
B. morisilkworm translation initiation factor (
BmTIF) was used as inner standard. The PCR amplification condition was 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 55°C for 30 s, 72°C for 30 s, and finally hold at 4°C. Three parallel experiments were performed. The result was expressed as ratio on
BmTIF(inner standard) mRNA, and the ratio of detected gene over
BmTIFin male silkworm was arbitrarily set as 1 (
Canbay et al. 2003 (link)
). The statistic significance of difference was analyzed with one sample
t-test.
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was synthesized using random primers and the PrimeScript RT Reagent Kit (Takara, China). Real-time polymerase chain reaction (qPCR) was performed using SYBR Premix Ex Tag (Takara, China). The PCR conditions were as follows: 95 °C for 15 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. β-actin was used as the internal control. The primer sequences for real-time PCR are listed in Table 1
4
qRT-PCR Analysis of GmNPR1 Gene Expression
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Total RNA was isolated using RNA isoreagent (TaKaRa Bio Inc., Shinga, Japan) according to the manufacturer's protocol. cDNA was synthesized from 1 μg total RNA in a 10 μL aliquot using Moloney Murine Leukemia Virus reverse transcriptase (M-MLV, TaKaRa Bio Inc.) at 42°C for 1 h with the oligo-dTadaptor primer (TaKaRa Bio Inc.) following the manufacturer's protocol. Real-time quantitative PCR was performed using 0.5 μL of reverse transcription production in a 25-μL reaction volume with SYBR Premix Ex Tag (TaKaRa Bio Inc.) on an ABIPRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using gene-specific primers: β-tubulin was used as a house-keeping gene according to Xu et al. (2010 (link)), and the GmNPR1 Gene-Bank accession number is FJ418595 ; upstream primer 5′-ATGGCAAGGTTGGATATGAAGC-3′ and downstream primer 5′-TGGCAGGTCTACACGCATCA-3′; the amplification size of the product was 132 bp. Each sample was run in triplicate. The relative transcript abundance was calculated using the 2−ΔΔCT method.
5
Comprehensive RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from cell lines and tissue samples using the TRIzol kit (Invitrogen, Carlsbad, CA, USA). RNA (1 μg) was reverse-transcribed into cDNA immediately using Prime-Script RT kit (Takara, Shiga, Japan) following manufacturer’s instructions. qRT-PCR was carried out with SYBR Premix EX Tag (Takara) on an ABI Prism 7500 fast RT-PCR instrument (Applied Biosystems, Foster City, CA). Each experiment was performed in triplicate. β-actin was used as the internal reference gene. Data were acquired during the extension step. Objective CT values were normalized to β-actin and 2-△Ct method was used to calculate relative mRNA levels of gene expression. Primer sequences for qRT-PCR were listed in Additional file 2 : Table S2. The qRT-PCR primers for COL4A1 are not exon spanning type, but their specificity has been tested.
6
Quantitative Analysis of SOX4 Expression
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Total RNA was extracted from the right lung lobes using TRIzol reagent (Takara Biotechnology Co., Kyoto, Japan) according to the manufacturer's instructions. Following purification according to the kit instructions, cDNA was reverse-transcribed from 1 μg RNA from each sample using Prime Script RT reagent kit with gDNA Eraser (Takara Biotechnology Co.). The primers were designed as follows: SOX4 forward, 5′-ATGTCCCTGGGCAGTTTCAG-3′ and reverse, 5′-TGCAATAGTCCGGGAACTCG-3′; GAPDH forward, 5′-AGACAGCCGCATCTTCTTGT-3′ and reverse, 5′-CTTGCCGTGGGTAGAGTCAT-3′. qPCR was performed using SYBR Premix Ex Tag (Takara Biotechnology Co.) on LightCycler (Thermo Fischer Scientific, Carlsbad, CA, USA); the amplification program was 95°C for 30 sec, 40 cycles of 95°C for 30 sec and 60°C for 34 sec. Relative mRNA expression of SOX4 normalized to GAPDH was determined by the 2−ΔΔCq method.
7
Quantitative Gene Expression Analysis in Strawberry
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Before RNA extraction, the achenes were stripped from the frozen strawberry fruit, and only the de-seeded flesh was processed for RNA isolation. RNA was isolated from either the deseeded flesh or seedlings by a modified CTAB method. After DNase I treatment, RNAs were used for cDNA synthesis by using the Primerscript RT reagent Kit with gDNA Erase (Takara). The cDNAs were used as templates for quantitative RT-PCR to measure the abundance of a certain transcript. Quantitative RT-PCR was performed using SYBR Premix Ex Tag (Takara) on a Bio-rad iQ5. Primers used are listed in Supplementary Table S6 . Results were analyzed by using the ΔΔCT method42 (link) using GAPDH as the control locus43 (link). Three biological and three technical replicates were performed and analyzed.
8
Transcriptional Profiling of Stress-Responsive Plant Genes
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The RNA of stress-treated seedlings was isolated using a TaKaRa MiniBEST Plant RNA Extraction Kit. Nine FveERFs from all the lineage-specific tandem repeats (all six genes from two tandem repeats plus three genes randomly selected from the six-gene tandem repeat, mrna08071–mrna08075 and mrna08077, Table S1 Table S1
9
Quantification of mESC Transcripts and Chromatin
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Total RNAs were extracted from mESCs (E14 and ZHBTc4) with TRIzol Reagent (Invitrogen). Extracted RNAs were synthesized into cDNAs by reverse-transcription with AMV Reverse Transcriptase for RT PCR analysis. RT PCR for cDNA was performed using SYBR premix Ex Tag (Takara) and normalized to 18S rRNA. For ChIP assays, SYBR® Green qPCR mix (Finnzymes, F-410) was used and the results are normalized to 1% input chromatin on CFX Connect Real-time PCR Detection System (Bio-Rad).
10
Isolation and Quantification of Plant RNA
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A modified CTAB method was used for RNA isolation from all the samples mentioned above. The isolated RNAs were treated with DNase I and used for cDNA synthesis using the Primerscript RT Reagent Kit with gDNA Eraser (Takara). qRT-PCR was performed using SYBR Premix Ex Tag (Takara) with the cDNA as the template. The IDT website (http://sg.idtdna.com/site ) was used to design the qRT-PCR primers for the FveIPT genes (Table S3 ). The sequence similarity between FveIPT3 and FveIPT4 is >90%; thus, we used a single primer set to investigate the combined expression of both genes. The results were analyzed using the −ΔΔCT method61 (link) with GAPDH62 (link) as the control locus. Three biological and three technical replicates were analyzed.
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