Total RNA was extracted using Trizol reagent (TAKARA, 9108), followed by Chloroform centrifugation and isopropanol precipitation. CDNA was synthesized using Primescript RT reagent Kit (Roche, 04896866001). Q-PCR was performed using the SYBR Green II PCR Mix (TAKARA) and the fluorescence ration PCR instrument IQ5 (Bio-Rad). The qRT-PCR primers used were listed in Additional file 2 : Table S1.
Sybr green 2 pcr mix
Manufactured by Takara Bio
Sourced in United States, China, Japan
SYBR Green II PCR Mix is a pre-formulated solution containing all the necessary components for real-time PCR amplification, including SYBR Green II dye, DNA polymerase, dNTPs, and buffer. It is designed for sensitive and specific detection and quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (3 mentions)
Trizol regent, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Roche (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Lightcycler 480 real time detection system, by Roche (1 mentions)
Trizol reagent, by Solarbio (1 mentions)
Primescript rt regent kit with gdna eraser, by Takara Bio (1 mentions)
Light cycler96 real time detection system, by Roche (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 thermocycler, by Roche (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
8 protocols using sybr green 2 pcr mix
1
Total RNA Extraction and qRT-PCR Analysis
2
Comprehensive RNA Extraction and qPCR Analysis
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Cells were lysed with Trizol regent (TAKARA). Total RNA was isolated by chloroform followed by precipitating with isopropanol. cDNA was synthesized with the PrimeScript™ RT reagent Kit (TAKARA) following the manufactory’s instructions. Primers designed and synthesized for RT-qPCR were listed in Supplementary Table 1 . Quantitative PCR was performed using the SYBR Green II PCR Mix (TAKARA) and the IQ5 (Bio-Rad).
3
RNA Isolation and RT-qPCR Analysis
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Cells were lysed with Trizol regent (TAKARA). Total RNA was isolated by chloroform followed by precipitating with isopropanol. cDNA was synthesized with the PrimeScriptTM RT reagent Kit (TAKARA) following the manufactory’s instructions. Primers designed and synthesized for RT-qPCR were listed in Supplementary Table S1 . Quantitative PCR was performed using the SYBR Green II PCR Mix (TAKARA) and the IQ5 (Bio-Rad).
4
Quantitative Real-Time PCR for Gene Expression
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RNA was extracted from the cell or tissues with Trizol (Invitrogen) according to the manufacturer's protocol. RNA samples were subjected to reverse transcription using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Real-time PCR was performed with SYBR Green II PCR Mix (Takara) using an IQ5 (Bio-Rad, Berkeley, CA, USA). Reactions were run in triplicate in three independent experiments. The primer sequences are provided in Supplementary Table S1 . Expression data were normalized to the geometric mean of housekeeping gene Gapdh to control the variability in expression levels and were analyzed using the 2−ΔΔCT method.
5
Quantifying Endometrial Gene Expression
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Total RNA was extracted from endometrial tissue and cultured endometrial epithelial cells using TRIzol reagent (Solarbio, Beijing, China) and was reverse-transcribed using the Prime Script RT Reagent Kit with a cDNA Eraser (Takara Bio, Dalian, China). mRNA levels were determined via qRT-PCR using the LightCycler 480 Real-Time Detection System (Roche, Basel, Switzerland). The reaction mixtures consisted of 10 μL of 2 × SYBR Green II PCR Mix (Takara Bio), 25 μmol/L each of forward and reverse primers, 2 μL of template, and distilled water up to a final volume of 20 μL. The reaction conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 10 s. A melting curve was obtained from 65 °C to 95 °C, increasing at intervals of 0.5 °C every 5 s. β-actin was used as an internal control. Four replicates were used for each sample to ensure the accuracy of the relative expression of target genes. The primers used were designed based on the mRNA sequence data available in GenBank; TLR4 [50 ], NFκB, TNF-α, Erα [51 (link),52 (link)], ERβ, PGR [53 (link)], β-actin [52 (link)], and the sequences are listed in Table 1 . The relative expression of target genes was calculated using the 2−ΔΔCT method [54 (link)].
6
Quantifying Gene and MicroRNA Expression in Spermatogenesis
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RNA was extracted from the isolated spermatogonia, pachytene spermatocytes, round spermatids, spermatozoa with TRIzol (Invitrogen) according to the manufacturer's protocol. For each sample, total RNA was used for reverse transcription using PrimeScript RT regent kit with gDNA Eraser (Takara). The quality of the resultant cDNAs was verified by PCR analysis of β-actin expression. The primer information is shown in the Supplementary Table 1 (see section on supplementary data given at the end of this article). qRT-PCR was performed with SYBR Green II PCR Mix (Takara) using an IQ-5 (Bio-Rad). The relative gene expression level was normalized to β-actin and analyzed using the 2 -ΔΔCT method. For real-time PCR of miRNA, stem-looped primers (Supplementary Table 2) were used for reverse transcription as described previously (Tang et al. 2006) (link). The relative expression level of miRNAs was normalized to 5s RNA and calculated using the comparative Ct method (2 -ΔΔCT ).
7
Quantitative Real-Time PCR Analysis
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Primers were selected according to the RNA-seq results (Supplementary Table S2 ). The LightCycler® 96 Realtime Detection System (Roche, Beijing, China) was used to detect and analyze the selected genes through quantitative Real-Time PCR (qRT-PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene. The reaction system was 20 μL and comprised 10 μL 2× SYBR Green II PCR Mix (Takara Bio, Beijing, China), 1 μL cDNA (25 ng), 1 μL primers, and 8 µL nuclease-free water. The reaction conditions were as follows: 95 °C for 300 s, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 2 min. A melting curve was obtained from 65 °C to 95 °C, which was increasing in increments of 0.5 °C every 5 s. The 2−∆∆Ct method was employed for calculation of relative expression.
8
Bovine Immune Gene Expression Analysis
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Total RNA was isolated from the thymus using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reversely transcribed to single-stranded cDNA using a reverse transcription kit (MBI Fermentas, Burlington, ON, Canada) according to the manufacturer's instructions. The qRT-PCR primers were designed according to the Bos grunniens CD3ε, CD79α, IgA, IgG, SIRPα, CD68 and β-actin gene sequences (GenBank accession numbers: KY911279, KY911280, MG432919, MF099643, MH347358, KY921959 and DQ838049, respectively) using Primer 5 software and synthesized by the Beijing Genomics Institute BGI Company (China). The qRT-PCR primer sequences are presented in Table 1. qRT-PCR was conducted with a Light-Cycler480 thermocycler (Roche, Manheim, Germany) in a 20 μL reaction volume consisting of 1 μL cDNA, 1 μL forward primer, 1 μL reverse primer, 10 μL 2×SYBR Green II PCR mix (TaKaRa, Shiga, Japan), 0.4 μL Rox, and 6.4 μL nuclease-free H 2 O. The RT-PCR conditions were as follows: one cycle at 95°C for 3 min, followed by 42 cycles of 95°C for 10 s, 60°C for 15 s and 72°C for 15 s. Four replicates were set for each sample to ensure the accuracy of the relative expression of the target genes in the sample. After amplification, according to the system-generated Ct value, the 2 -ΔΔ Ct method was used with β-actin as an internal standard to normalize the amount and quality of each cDNA.
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