The expressions of granzyme B in the spleen and lymph node were investigated by RT-qPCR analysis. Briefly, total RNA was isolated from cells. Reverse transcription was performed using PrimeScript Reverse Transcriptase kit (Takara) and cDNA was used for subsequent real-time PCR reactions. Quantitative real-time PCR was conducted on an ABI Prism 7500 instrument using the Maxima SYBR green qPCR Master Mix (Takara). The cycling parameters were 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s; each assay was performed in triplicate, and the relative expression levels (defined as fold changes) of the target genes were normalized. The following primers were used: β-actin (sc-108070-PR) and granzyme B (sc-35508-PR) (Santa Cruz). However, primer sequences are not provided by Santa Cruz, as stated in their datasheets: “semi-quantitative RT-PCR may be performed to monitor gene expression knockdown using RT-PCR primer: β-actin (m)-PR: sc-108070-PR (600 bp) and granzyme B (m)-PR: sc-35508-PR (536 bp)”.
Maxima sybr green qpcr master mix
Manufactured by Takara Bio
Sourced in Japan
Maxima SYBR Green qPCR Master Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) analysis. It contains all the necessary components for efficient DNA amplification and detection, including a hot-start Taq DNA polymerase, SYBR Green I dye, and optimized buffer system.
Automatically generated - may contain errors
Lab products found in correlation
β actin sc 108070 pr, by Santa Cruz Biotechnology (2 mentions)
Primescript reverse transcriptase kit, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Granzyme b, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Takara Bio (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Maxima first strand cdna synthesis kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rr036a, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
8 protocols using maxima sybr green qpcr master mix
1
Granzyme B Expression Analysis in Spleen and Lymph Node
2
EDARADD mRNA Expression in TSCC Cell Lines
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Total RNA was isolated from the TSCC cell lines and purified using SuPerfecTRI™ reagent (Shanghai Pufei Biotechnology Co., Ltd.) according to the manufacturer's instructions. The mRNA fragments were used to generate cDNAs using M-MLV reverse transcriptase (Promega Corp.) for 60 min at 37˚C according to the manufacturer's protocol. The abundance of the EDARADD mRNA in the CAL27, SCC25 and SCC9 cells was then detected by qPCR using the Maxima SYBR Green qPCR Master Mix (Takara Bio, Inc.) by a two-step method. The cycling conditions were as follows: 95˚C for 10 min, then 40 cycles at 95˚C for 15 sec and 60˚C for 60 sec. The reverse transcription primer and microRNA PCR primers were purchased from Guangzhou Ruibo Biotechnology Co., Ltd. The relative change in the mRNA expression levels was calculated by the comparative threshold cycle method (2-ΔΔCq) (22 (link)) using the GAPDH as an internal reference gene control. The experiment was repeated three times. The sequences of the primers used are as follows: EDARADD forward, 5'-GACCAACCCAAAGAGGACAG-3' and reverse, 5'-CCAGAATGATGAGGCACCAT-3'; GAPDH forward, 5'-TGACTTCAACAGCGACACCCA-3' and reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'.
3
Quantitative Real-Time PCR for Gene Expression
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Total RNA was extracted with the TRIzol reagent (Invitrogen), and reverse-transcribed into cDNA templates using the PrimeScriptTM RT reagent kit (Takara, Japan) following the manufacturer’s instruction. Gene expression levels were quantified using the Maxima SYBR Green qPCR Master Mix (Takara) on the StepOne Plus Real-Time PCR System (Applied Biosystems Inc., Foster City, CA). Sequences of primers were listed in Supplementary Table 1 .
4
Quantifying DACT1 Expression in Cells
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Total RNA was isolated from cell lines and tissues using TRIZOL (TAKARA, Japan) according to the manufacturer’s protocol, and DACT1 expression was assessed by quantitative Real-time RT-PCR using the following primers: sense, 5′-GACGAGCAGAGCAATTACACC-3′; antisense, 5′-ACCGTTTGAATGGGCAGA-3′. DACT1 expression was normalized to β-actin expression using the comparative CT-method47 (link) with the following primers: sense, 5′-CCTGTGGCATCCACGAAACT-3′; antisense, 5′-GAAGCATTTGCGGTGGACGAT-3′. All reactions were performed in triplicate with the Maxima SYBR Green qPCR Master Mix (TAKARA, Japan) in 10 μl with 5 μl SYBR, 0.4 μl of each primer (10 μM), 3.2 μl DEPC treated water and 1000 ng cDNA, using the Bio-RAD CFX96 Real-Time System at 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 58 °C for 30 s and 72 °C for 15 s.
5
Quantitative Reverse Transcription PCR of hiPSC-Heps
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Total RNA was extracted from hiPSC-Heps using TRIzol™ reagent (15596026, Invitrogen, Carlsbad, CA, USA). After RNA extraction, the concentration and purity of the RNA were measured using a spectrophotometer (Nanodrop 2000, Thermo Scientific, Waltham, MA, USA). Reverse transcription of RNA was performed using PrimeScript™ RT Master Mix (6210 A, TaKaRa, Kyoto, Japan), following the manufacturer’s protocol. The qRT-PCR reaction system was then prepared using Maxima SYBR Green qPCR Master Mix (RR420L, TaKaRa). Data collection was performed using a real-time PCR detection system (7500, Applied Biosystems, Carlsbad, CA, USA). The relative gene expression levels were calculated using the 2–DDCt method with b-actin selected as the housekeeping gene. Primers used for qRT-PCR are listed in Table A.1
6
Chondrocyte RNA Extraction and qPCR Analysis
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The cartilaginous tissues or primary cultured chondrocytes from mice were collected and lysed in TRIzol (Invitrogen) for RNA isolation according to the manufacturer’s standard protocol. cDNA was synthesized from 1 μg RNA Maxima First Strand cDNA Synthesis kit (Takara). Real-time PCR was performed on ABI Fast7500 with Maxima SYBR Green qPCR Master Mix (Takara). The primer pairs have been previously described25 (link),55 (link)–58 (link) and are included in Supplementary Table 3 . Fluorescence qPCR was performed by real-time fluorescence qPCR instrument (qTOWER3G, Jena Bioscience) and its application software (qPCRsoft 3.4). Real-time PCR results were analysed by Microsoft Excel (2306 Build 16.0.16529.20164).
7
Quantifying Immune Receptor Expression in DCs
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The expressiones of MR, TLR4 in DCs were investigated by RT-qPCR analysis. Briefly, total RNA was isolated from cells. Reverse transcription was performed using PrimeScript Reverse Transcriptase kit (Takara) and cDNA was used for subsequent real-time PCR reactions. Quantitative real-time PCR was conducted on an ABI Prism 7500 instrument using the Maxima SYBR green qPCR Master Mix (Takara). The cycling parameters were 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 34 s; Each assay was performed in triplicate, and the relative expression levels (defined as fold changes) of the target genes were normalized. The following primers were used (Santa Cruz): β-actin (sc-108070-PR), MR (sc-45361-PR), TLR4 (sc-40261-PR), MyD88 (sc-35987-PR) and IRAK4 (sc-45401-PR). However, primer sequences are not provided by Santa Cruz, as stated in their datasheets: “Semi-quantitative RT-PCR may be performed to monitor gene expression knockdown using RT-PCR Primer: β-Actin (m)-PR: sc-108070-PR (600 bp); CD206 (m)-PR: sc-45361-PR (498 bp) ; TLR4 (m)-PR: sc-40261-PR (434 bp); MyD88 (m)-PR: sc-35987-PR (545 bp); IRAK-4 (m)-PR: sc-45401-PR (491 bp)”.
8
Liver RNA Isolation and Quantification
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Total RNA was isolated and extracted from the liver tissues using TRIzol reagent (Takara, Otsu, Japan). It was transcribed into cDNA using the reverse transcription kit (RR036A, Takara, Otsu, Japan) according to the manufacturer’s protocols. The relative mRNA levels were evaluated by quantitative PCR using Maxima SYBR Green qPCR Master Mix (Takara, Otsu, Japan). The primers used for real-time quantitative PCR are listed in Additional file 2 : Table S1.
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