Total RNA was extracted from the testis tissue using an RNApure total RNA isolation kit (Bioteke, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was obtained from 5 μL of the total RNA using an AMV First Strand cDNA Synthesis Kit (QiaGEN, Shanghai, China). Amplification reactions were performed in a total volume of 25 μL of PCR mixture from the SYBR Green I Real Time PCR KIT (Takara, Beijing, China) containing 5 μL 5× PrimeSTAR™ buffer (Mg2+ plus), 2 μL dNTPs (2.5 mM each), 0.25 μL PrimeSTAR™ HS DNA polymerase (2.5 U/μL), 0.5 μL first-strand cDNA, 0.5 μL (20 pmol) each of the specific primers for Vasa, SMAD1, Stella, Dazl, GCNF, c-kit, and β-actin (as reference) (for primer sequences see Table 2 ), and 16.25 μL RNase-free water. The samples were denatured at 94 °C for 3 min, followed by 40 amplification cycles of 94 °C for 30 s, 50, 51, 56, 58, 55, 58 and 56 °C (for Vasa, SMAD1, Stella, Dazl, GCNF, c-kit, and β-actin respectively) for 15 s, and 72 °C for 30 s, in a thermal cycler (Roche, Basel, Switzerland); fluorescence signal intensity was measured at 72 °C during each cycle. The PCR products were identified by melting curves: 95 °C for 2 min, 72 °C for 1 min, 95 °C for 30 s with steps of 0.5 °C/s, 30 °C for 1 min. Each product represented a single peak.
Sybr green 1 real time pcr kit
Manufactured by Takara Bio
Sourced in Japan, United States
The SYBR Green I Real-Time PCR kit is a laboratory product designed for quantitative real-time PCR (qRT-PCR) analysis. It contains the necessary reagents, including SYBR Green I dye, for the detection and quantification of DNA sequences during the PCR amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Thermal cycler, by Roche (1 mentions)
Rnapure total rna isolation kit, by BioTeke (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Cfx96 thermal cycler, by Bio-Rad (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Takara Bio (1 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (1 mentions)
One step primescript mirna cdna synthesis kit, by Takara Bio (1 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Midetect a track mirna qrt pcr starter kit, by RiboBio (1 mentions)
First strand cdna synthesis kit, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Cellamp whole transcriptome amplification kit, by Takara Bio (1 mentions)
Minioption thermocycler, by Bio-Rad (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using sybr green 1 real time pcr kit
1
Gene Expression Analysis in Testis Tissue
2
Quantifying miR-423-5p expression in human samples
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Human colon samples and transfected cells were lysed using TRIzol® reagent (Thermo Fisher Scientific, Inc.) and total RNA was extracted following the manufacturer's protocol. Reverse transcription was performed on the isolated total RNA using a PrimeScript® RT Master Mix kit (Perfect Real Time; cat. no. RR036A; Takara Bio, Inc., Kusatsu, Japan) and qPCR was performed using a SYBR Green I Real Time PCR kit (cat. no. RR420A; Takara Bio, Inc.), according to the manufacturer's protocol. Reverse transcription was performed at 65°C for 5 min, 30°C for 10 min, 42°C for 10 min and 2°C for 3 min. qPCR conditions were as follows: Denaturation at 94°C for 2 min, amplification for 30 cycles at 94°C for 30 sec, annealing at 59°C for 30 sec and extension at 72°C for 1 min, followed by a terminal elongation step at 72°C for 10 min. The primers for miR-423-5p were purchased from Guangzhou RiboBio Co., Ltd. (cat no. S170721170309; Guangzhou, China). Sequences were not supplied due to the rules of the company. U6 was used as an internal control. qPCR analysis was performed using a Bio-Rad CFX96 thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Data were quantified using the 2−ΔΔCq method (11 (link),12 (link)).
3
Quantification of miRNA Expression
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Total RNA was extracted from the tissues and cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. The miRNAs were detected by real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The cDNAs were produced by reverse transcription kits (Takara, Dalian, China) and miRNA-specific bulge-loop™ miRNA RT primers (Ribobio Co, Guangzhou, China) (Table S2 ). We performed qRT-PCR by a StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) and an SYBR Green I Real-Time PCR kit (TaKara, Dalian, China). We used the comparative delta CT (2−∆∆Ct) method to estimate the relative expressions of miR-29a. U6 small nuclear 2 (RNU6) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as endogenous controls in this study.
4
miR-124 and DLL4 Expression Analysis
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Total RNA was extracted from NSCs using TRIzol reagent (invitrogen) based on the instruction specified by the manufacture. For miR-124 expression detection, aliquots of total RNA (0.5 μg) was reversely transcribed into cDNA using the one-step Primescript miRNA cDNA synthesis kit (Takara, Dalian, China) and RT-PCR was performed using the TaqMan MicroRNA Assay kit (Applied Biosystems Inc., Foster City, CA, USA). For DLL4 mRNA expression evaluation, the first strand of cDNA was synthesized from total RNA using M-MLV reverse transcriptase (Clontech, Palo Alto, CA, USA) and RT-PCR was carried out by the SYBR Green I Real-Time PCR kit (Takara). Real-time PCR was then executed with an ABI7300 Real-Time PCR System (Applied Biosystems Inc.). Relative gene expression levels of miR-124 and DLL4 were calculated using the 2−ΔΔCt method, with U6 and GAPDH as respective internal control.
5
Quantifying Peg13, Sox13, and miRNA Levels
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After repeated exposure of NSCs to sevoflurane, the relative levels of Peg13, Sox13 mRNA and relevant miRNA transcripts to U6 or GAPDH in different groups of cells were quantified by qRT-PCR using specific primers, the SYBR Green I Real-Time PCR kit (Takara), or miDETECT A TrackTM miRNA qRT-PCR Starter Kit (RIBOBIO), respectively. The data were analyzed by the 2−ΔΔCt method. The sequences of primers were Peg13 forward 5'-CTCACTTTGGTTTGAATGGGAT-3' , and reverse 5'-AAGACGATTAGATTGGGTTGC-3'; Sox13 forward 5'-AGCAAGATCCTTGGTTCTCG-3' , and reverse 5'-GGAGACTGCAGGTATTGATG-3' ; GAPDH forward 5'-AATGGATTTGGACGCATTGGT-3' and reverse 5'-TTTGCACTGGTACGTGTTGAT-3' . The primers for miRNA and U6 were purchased from RIBOBIO.
6
Quantifying Gene Expression in Glioma Cell Lines
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Total RNA was extracted from U251 and U87 cell lines using Trizol (Takara, Japan) and cDNA was synthesized using the cDNA first-strand synthesis kit (Takara, Japan) following the manufacturer’s protocols. QRT-PCR was performed to measure gene expression with the Takara SYBR Green I Real-Time PCR Kit following the manufacturer’s instructions (Takara, Shiga, Japan). Following primers were used for CUX1, forward primer: 5′-CGCCAAAAACAGCACACTCA-3′; reverse primer: 5′-CCGACTTTCAGGCTGGTCTT-3′; for β-catenin, the forward primer was 5′-GGAGGAAGGTCTGAGGAGCA-3′ and reverse primer was 5′-CCAGTGACTAACAGCCGCTT-3′. For GAPDH, the forward primer was 5′-ATCTGTTTCACAGTCTGGGAC-3′, and the reverse primer was 5′-CCTGCTTGTTGGCAAATACC-3′. The qRT-PCR cycle profile was performed at 95 °C for 10 min to activate DNA polymerase, followed by 45 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 10 s. The GAPDH was used as an internal reference, and the relative gene expression as fold change was calculated using the 2−ΔΔCT method. Each experiment was repeated three times, and data were presented as mean ± SD from three independent experiments.
7
Blastocyst Transcriptome Analysis Protocol
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Each group of blastocyst pool was separately collected and performed according to the Cell Amp Whole Transcriptome Amplification Kit (TaKaRa, Japan). cDNA was amplified through 20 cycles of PCR. Subsequently, 10-fold diluted cDNA was used as the template for Q-PCR, which was performed based on the SYBR Green I Real-Time PCR Kit (TaKaRa). The primers were presented in Supplementary Table S1. Each gene was tested in triplicate.
8
Quantifying Gene Expression via qRT-PCR
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The expression of PAR-1, SIRT1, and thrombin was measured by quantitative Real-Time PCR (qRT-PCR). TRIZOL reagent (Invitrogen, Grand Island, NY, USA) was applied to extract total RNA. Subsequently, the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to convert RNA into cDNA, and SYBR-Green I Real-Time PCR kit (TaKaRa Biotechnology, Dalian, China), with a Bio-Rad MiniOption thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA), was used for detection. Primer sequences were as follows:
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