Briefly, total RNA was isolated from tissues and cells by using TRIzol® reagent (Thermo Fisher Scientific, Inc.) and then reverse transcribed using a QuantiTect Reverse Transcription Kit (QIAGEN, Valencia, CA) according to the manufacturer’s specifications. qPCR amplification was performed by using SYBR-Green PCR mix (Takara), and the expression levels of target genes were normalized to the level of GAPDH. The primer sequences were as follows: HLA-DRB5 Forward Sequence-GAACAGCCAGAAGGACTTCCTG and Reverse Sequence-GCAGGATACACAGTCACCTTAGG. CCDC50 Forward Sequence-AGTGATGAACCTCACCATTCTAAG and Reverse Sequence-GAAATGCCGTGTGGAACTCTGC.
Sybr green pcr mix
Manufactured by Takara Bio
Sourced in Japan, United States, China, Germany
SYBR Green PCR mix is a reagent used in quantitative real-time PCR (qPCR) assays. It contains SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence upon binding. This allows for the monitoring and quantification of DNA amplification during the PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (41 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Trizol, by Takara Bio (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Takara Bio (5 mentions)
Primescript reverse transcriptase reagent kit, by Takara Bio (5 mentions)
Primescript rt master mix kit, by Takara Bio (4 mentions)
M mlv reverse transcriptase, by Promega (4 mentions)
Lightcycler 96, by Roche (4 mentions)
Reverse transcription kit, by Takara Bio (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Reverse transcription system kit, by Takara Bio (3 mentions)
Cdna synthesis kit, by Takara Bio (3 mentions)
Lightcycler 480, by Roche (3 mentions)
Abi prism 7500, by Thermo Fisher Scientific (3 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Cfx connect, by Bio-Rad (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Quick rna isolation kit, by Tsingke (2 mentions)
M mlv reverse transcriptase, by Tsingke (2 mentions)
99 protocols using sybr green pcr mix
1
Quantitative Transcriptional Analysis
2
Quantitative Real-Time PCR Protocol
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The total RNA of cells and tissues were extracted from Trizol (Cat#9109; TaKaRa, Japan), and the PrimeScript RT Master Mix kit was used to reverse it into cDNA (Cat#RR047A; Takara, Japan). All of the mRNA levels were determined by employing the SYBR Green PCR Mix (Cat#RR420A; Takara, Japan) in conjunction with the CFX Connect (Bio-Rad, USA) as previous study [44 (link)]. The 2−ΔΔCT approach was utilized for data analysis, and each experiment was carried out with three separate sets of controls. The primer sequences used in this work were all synthesized by Sango Biotech (Shanghai, China), and they were all listed in the Table 1 .
3
Quantitative Gene Expression Analysis in Bermudagrass
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For each treatment, three biological replicates were taken from different potted plants. Trizol reagent (Invitrogen, America) was used to isolated total RNA from about 0.1 g samples of leaves and roots. DNasel was used to remove contaminating genomic DNA from RNA. A UV spectrophotometry NanoDrop was used to examine the RNA concentration and purity (Thermo Fisher Scientific, Lenexa, Ks, USA). Using a Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR with genome-DNA-removing enzyme, 2.5 ug RNA was reverse transcribed to cDNA (Yesen, Nanjing, China). The qPCR was carried out on a Quant Studio 6 detection system (ABI, Forster City, CA, USA) with a SYBR green PCR mix (Takara, RR420A, Shika, Japan). The following was the real-time PCR program: 95°C for 5 minutes; 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Table 2
4
RNA Extraction and qRT-PCR Analysis
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In brief, total RNA was extracted from tissues or cells using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The concentration of total RNA was measured by Qubit 4.0. Then, 2 μg of isolated RNA was used for the reverse transcription reaction with the PrimeScript reverse transcriptase (RT) reagent kit (TaKaRa, Shiga, Japan). Finally, qRT–PCR was performed using SYBR green PCR mix (Takara). GAPDH or U6 served as internal controls.
5
Quantitative Analysis of NMDAR Subunits
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Total RNAs from tissues were extracted with TRIzol reagent according to the manufacturer’s manuals. The total RNA was first reverse transcribed to cDNA using PrimerScript™ RT reagent Kit with gDNA Eraser (Takara RR047A). SYBR Green PCR mix (Takara RR820A) was used for fluorescence PCR, in 10 μL of reaction mixture containing 4 μL cDNA and 1 μL (1 pmol/μL) primers with a protocol of 95°C for 2 min, then 95°C for 10 s and 56°C for 30 s. Protocols of step 2 and step 3 were repeated for 39 cycles. For RNA internal control, housekeeping gene ACTIN was used to normalize the mRNA expression of NMDAR subunits. The primers of all seven NMDAR subunits and ACTIN gene are shown in Table 2 . Results of the qPCR were represented as Ct values, and comparative ΔΔCT methods were used to calculate the relative mRNA expression of NMDAR subunits.
6
Quantitative Real-Time PCR Analysis in Plants
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Total RNA was extracted using the TRizol reagent. First-stand cDNA was synthesized by a PrimeScript First-strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Real-time PCR was performed in a Two-color Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR Green PCR Mix (TaKaRa, Dalian, China). The 18S rRNA and Actin were chosen as internal controls in sugar beet and Arabidopsis, respectively [36 (link)]. PCR reaction was carried out in 10 µL volumes using the following amplification protocol: 94 °C for 4 min; 94 °C for 30 s, 53 °C for 20 s, and 72 °C for 70 s; and 72 °C for 4 min and 45 cycles. The primers used for qRT-PCR analysis are listed in Table S1 . The primer sequence of SOD and POD genes for qRT-PCR were acquired from reference [30 (link)], and other primers were designed by Primer 5. A total of three biological replicates and three technical replicates were performed for the quantitative Real-Time PCR analyses.
7
Quantification of miRNA and mRNA Expression
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miRNA was prepared using the miRNeasy Mini kit (Qiagen, Inc.) and total RNA was prepared using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For miRNA reverse transcription, cDNA was synthesized using TaqMan® miRNA reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 42°C for 1 h. For mRNA reverse transcription, cDNA was synthesized using the Oligo dT primer (Takara Biotechnology Co., Ltd.) at 42°C for 1 h. qPCR was performed using an SYBR Green PCR mix (Takara Biotechnology Co., Ltd.). qPCR was conducted as follows: 95°C for 15 min, followed by 40 cycles of 94°C for 15 sec, 55°C for 30 sec and 70°C for 30 sec, and a final extension step at 72°C for 5 min. The following primers were used for RT-qPCR analysis: miR-21 forward (F), 5′-GCCCGCTAGCTTATCAGACTGATG-3′ and miR-21 reverse (R), 5′-CAGTGCAGGGTCCGAGGT-3′; U6 F, 5′-TGCGGGTGCTCGCTTCGCAGC-3′ and U6 R, 5′-CCAGTGCAGGGTCCGAGGT-3′; PTEN F, 5′-TTGGCGGTGTCATAATGTCT-3′ and PTEN R, 5′-GCAGAAAGACTTGAAGGCGTA-3′; GAPDH F, 5′-AGGTCGGTGTGAACGGATTTG-3′ and GAPDH R, 5′-TGTAGACCATGTAGTTGAGGTCA-3′ The RT-qPCR assays were performed in triplicate and the relative expression levels were calculated based on the 2−ΔΔCq method (20 (link)).
8
Circular RNA EIF3H Quantification
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Transcription was prevented by the addition of 2 μg/ml Actinomycin D (Sigma-Aldrich, USA) for the indicated time. Total RNA (2 μg) was incubated for 30 min at 37 °C with 3 U/μg of RNase R. After treatment with Actinomycin D and RNase R, the RNA expression levels of circEIF3H and EIF3H mRNA were detected by qRT-PCR using the SYBR green PCR mix (Takara).
9
RNA Extraction and qRT-PCR Protocol
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Total RNA was extracted from tissues using the miRNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer instructions. Total RNA containing small RNA was extracted from 500 μL plasma using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and the miRNeasy Mini kit according to the manufacturer instructions. cDNA was synthesized via M-MLV reverse transcriptase (Promega, Madison, WI, USA). Oligo (dT)15 was used as the RT primers for reverse transcription of mRNAs. MiR-221/222 and U6 snRNA were reverse transcribed using specific RT primers. Quantitative RT-PCR was performed in an Step-One real-time PCR System (Applied Biosystems, Foster City, CA, USA) using the SYBR Green PCR Mix (Takara, Dalian, China) at 95 °C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min according to the manufacturer's instructions. The comparative Ct method was used to quantify target genes relative to the endogenous control. For the mRNAs, data were normalized to the endogenous GAPDH or beta-actin control. For the miRNAs, U6 snRNA or cel-miR-39 was used as the endogenous control. All PCR reactions were performed in triplicate. The primers used for PCR are listed in Table S1 .
10
Quantitative Analysis of miRNA and mRNA
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Total RNA was isolated with Trizol (Sangon Biotech) following standard instruction. The miRNA cDNA library was established using SuperMixQuantiMir cDNA Kit (Transgen Biotec, Beijing, China) following reverse transcription of the RNAs using M-MLV RTase cDNA Synthesis Kit (TaKaRa, Dalian, China). SYBR Green PCR mix (TaKaRa) was used in qRT-PCR for quantification of miRNA and mRNA expression with U6 snRNA and 18S RNA as the internal controls, respectively. Relative quantification (2−ΔΔCT) was used for results analysis. PCR primers are listed in Supplementary Table S2 .
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