Fifty hearts were placed into 1 mL of Trizol reagent lysis solution for homogenization and RNA extraction. Trizol was used to extract the organic solvent, and 10 μg total RNA was purified using oligo (dT) synthesized from total RNA with superscript II reverse transcriptase (Invitrogen). qPCR amplification reactions were performed in triplicates by mixing 1 μL of RT product with 10 μL of SYBR qPCR Mastermix (TaKaRa) containing the appropriate PCR primers. Thermal cycling and fluorescence monitoring were performed in an ABI7300 (Applied Biosystems, United States) using the following PCR conditions: (30 s at 95 °C, 5 s at 95 °C, and 30 s at 60 °C) × 40. Values were normalized with rp49. Primers used were as follows: rp49(the reference gene)F: 5′-CTAAGCTAGTCGCACAAATAGG-3′R; 5′-AACTTCTTAGAATCCGGTAGGG-3′mtpF; 5-ACGGAAATCCAGCAGAACACT-3′R:5′-ATACGTAAAGCCAACGGCCA-3′Acox3F; 5′-ACTTCCGTAGCGGACCTTTAG-3′R:5-GCAGAAGATAGTAGGGGTTCCA-3Had1F; 5-CTAAATAGCAAGGTACGCGGC-3′R; 5-GATAGTAGGGCCGTAGCGATAA-3′.
Sybr qpcr master mix
Manufactured by Takara Bio
Sourced in Japan, United States
SYBR qPCR Master Mix is a ready-to-use solution for quantitative real-time PCR amplification and detection using SYBR Green I dye. It contains all the necessary components, including DNA polymerase, dNTPs, and SYBR Green I, to perform qPCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Abi 7300, by Thermo Fisher Scientific (4 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (3 mentions)
All in one mirna qrt pcr detection kit, by GeneCopoeia (2 mentions)
Total rna extraction kit, by BioFlux (2 mentions)
Paris kit, by Thermo Fisher Scientific (2 mentions)
Primesoript rt master mix, by Takara Bio (2 mentions)
Ez magna rip kit, by Merck Group (2 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Trizol reagent, by Sangon (1 mentions)
Cdna reverse transcription kit, by Takara Bio (1 mentions)
Real time pcr system, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Takara Bio (1 mentions)
Ncode mirna first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rotor gene 3000, by Qiagen (1 mentions)
Thermoscript rt pcr system, by Thermo Fisher Scientific (1 mentions)
23 protocols using sybr qpcr master mix
1
Cardiac RNA Extraction and qPCR Analysis
2
Quantitative RNA Expression Analysis
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Extraction of total RNA from lung tissue and cells was performed with TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and further reverse-transcribed using 5× Prime Script RT Master Mix kit (Takara, Kusatsu, Japan). SYBR qPCR Master Mix (Takara) was used to carry out quantitative PCR according to the manufacturer’s instructions. Target gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels in respective samples as an internal control and calculated using the 2−ΔΔCt method. The primer sequences are shown in Table 1 .
3
Total RNA Extraction and qPCR Analysis
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Total RNA from HCC cells or tissues was extracted using total RNA extraction kit (BioFlux, Hangzhou, China) according to the manufacturer’s instructions. For nuclear/cytoplasmic separation assay, cytoplasmic and nuclear RNA were separately isolated using PARIS kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol. The RNA was reversely transcribed to first-strand cDNA using PrimeScript RT reagent kit (Takara, Dalian, China). qPCR was performed with SYBR qPCR master mix (Takara), taking GAPDH as the internal reference. Reverse transcription and qPCR of miRNAs were conducted with All-in-One miRNA qRT-PCR detection kit (GeneCopoeia, Guangzhou, China), taking U6 RNA as the internal control. The primers were listed in Supplementary Table 1 .
4
Quantifying Gene Expression Changes in TAD Reorganization
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RNA extraction and reverse transcription Total RNA was extracted from cell pellets using RNAzol reagent (MRC) and cDNA was synthesized using Primesoript RT Master Mix (Takara).
Real time qPCR qPCR was performed using SYBR qPCR Master Mix on LightCycler 480 II system.
Data analysis The fold change (FC) of experimental group versus control group was calculated. △Ct was calculated as △Ct = Ct (test gene) – Ct (Ref. gene). △△Ct was calculated as △△Ct = △Ct (experimental group) – △Ct (control group). The FC of a test gene in experimental group versus control group was calculated as FC = 2ˆ(-△△Ct). Each gene tested in triplicates in every independent experiment, and all experiments were triplicated.
5
RNA Extraction and RT-qPCR Analysis
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TRIzol® reagent (500 µl) (Sangon Biotech Co., Ltd.) was used to extract the total RNA, and 500 ng RNA were used to synthesize cDNA for reverse transcription using the 5X Prime Script RT Master Mix (Takara Bio, Inc.). The cDNA (1 µl) was used as the template for RT-qPCR using SYBR qPCR Master Mix (Takara Bio, Inc.) according to the manufacturer's protocol. The PCR reaction conditions were 95°C for 5 min, 95°C for 30 cycles, 60°C for 30 sec, 72°C for 30 sec, and finally 72°C for 7 min. The expression levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and analyzed using the 2−ΔΔCq (30 (link)) method. The primer sequences used are presented in Table II .
6
Quantitative Analysis of Glioma Gene Expression
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First, RNA from glioma and paracancerous tissues was extracted using Trizol (Invitrogen, USA) extraction reagents. Isolated RNA was converted into cDNA via a cDNA reverse transcription kit (TaKaRa, Cat: RR047A, Japan). Following this, the relative expression of cDNA was visualized using the SYBR qPCR Master Mix (Takara, Cat: RR820A, Japan) on a Real-Time PCR system (Applied Biosystems, Foster City, USA). β-Actin was used as a reference gene, and relative gene expression was quantified using the formula: 2–ΔΔCt.
7
RNA Extraction and qPCR Analysis of LUAD Tissue
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Total RNA was harvested from LUAD tissue using Trizol and reverse-transcribed into cDNA using PrimeScript RT reagent kit (with gDNA Eraser) (Takara). The cDNA was amplified using the SYBR qPCR Master Mix (Takara) on a real-time PCR system (LightCycler480). Primer sequences are shown in Table 2 . All primers were synthesized by Sangon Biotech Co. Ltd. (Shanghai). RT-PCR was carried out as previously reported [15 (link)].
8
Quantitative Detection of miR-27a and Genes
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Total RNA from LX2 cells was extracted using Trizol reagent (Invitrogen). cDNAs and the first-strand cDNAs of miRNA were produced according to the manufacturer’s instructions for Thermoscript RT-PCR system (Invitrogen) or NCode miRNA First-Strand cDNA Synthesis kits (Invitrogen). For the quantitative detection of miR-27a and mRNAs of interested genes, the templates and primer sets (Table S1 ) were mixed with SYBR qPCR master mix (TaKaRa, Dalian, China), and real-time PCR was performed using Rotor-Gene 3000 (Corbett Research, Sydney). The cycling parameters were: initial denaturing at 94°C for 15 sec, followed by 40 cycles of 94°C denaturing for 10 sec, primer annealing and extension at 60°C for 40 sec. To ensure the specificity of the amplification reaction, melting curve analysis was performed. The expression of miR-27a was normalized to U6snRNA, and mRNAs were normalized to GAPDH. Relative gene expression was presented by comparative CT method.
9
Evaluating Pluripotency Gene Expression
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RNA extraction and reverse transcription Total RNA was extracted from cell pellets using RNAzol reagent (MRC) and cDNA was synthesized using Primesoript RT Master Mix (Takara).
qPCR qPCR was performed using SYBR qPCR Master Mix on LightCycler 480 II system.
Data analysis The fold change (FC) of experimental group versus control group was calculated. Ct was calculated with Ct = Ct (test gene) - Ct (Ref. gene). Ct was calculated with Ct = Ct (mutant or rescue group) – Ct (wide type group). The FC of a test gene in experimental group versus control group was calculated with FC = 2ˆ(-Ct). Each gene tested in triplicates in every independent experiment, and all experiments were triplicated.
10
RNA Immunoprecipitation Assay Protocol
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RNA Immunoprecipitation (RIP) assay was performed using EZ-Magna RIP kit (Millipore/Merck, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, the cells were lysed and incubated with antibody-coated beads at 4°C overnight. Subsequently, the co-immunoprecipitates were treated with proteinase K at 55°C for 30 min. RNA was purified with phenol:chloroform:isoamyl alcohol (125:24:1), precipitated with ethanol overnight, and reversely transcribed into cDNA using PrimeScript RT reagent kit (Takara). Then the cDNA was analyzed by qPCR with SYBR qPCR master mix (Takara).
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