Total RNAs were isolated from cells by using Trizol reagent (Life Technologies, Rockville, MD, USA) according to the manufacturer’s protocol. Total RNA was quantified by spectrophotometry. One microgram of total RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) and following manufacturer’s protocol. Real-time PCR was performed on an applied Biosystems StepOnePlus™ Real-Time PCR System (Applied Biosystems; Life Technologies, Carlsbad, CA, U.S.A.) using SYBR® Green Real-Time PCR Master Mix (Life Technologies), and relative quantification (RQ) was calculated by using StepOne™ software V2.2.2, based on the equation RQ = 2−ΔΔCt, where Ct is the threshold cycle to detect fluorescence. Ct data were normalized to the internal standard RPLP0. Primer sequences were as follow: iNOS; Sense: GTT CTC AGC CCA ACA ATA CAA GA, Anti-sense: GTG GAC GGG TCG ATG TCA C; RPLP0: Sense: GGG CAT CAC CAC GAA AAT CTC, Anti-sense: CTG CCG TTG TCA AAC ACC T.
Sybr green real time pcr master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, Germany, China, Italy
SYBR Green Real-Time PCR Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains SYBR Green I dye, DNA polymerase, dNTPs, and necessary buffer components for performing real-time PCR reactions.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (126 mentions)
Trizol, by Thermo Fisher Scientific (40 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (27 mentions)
Rneasy mini kit, by Qiagen (23 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (19 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (16 mentions)
Primescript rt reagent kit, by Takara Bio (14 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (10 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (10 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (10 mentions)
Dnase 1, by Thermo Fisher Scientific (9 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (8 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (8 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (7 mentions)
M mlv reverse transcriptase, by Promega (7 mentions)
Superscript 3, by Thermo Fisher Scientific (7 mentions)
Rneasy kit, by Qiagen (7 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
350 protocols using sybr green real time pcr master mix
1
Quantitative Analysis of iNOS Gene Expression
2
Quantifying miR-1207-3p Expression in Prostate Cancer
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Prostate tumor tissues were collected at prostatectomy from 404 patients with primary PCa. After histopathologic confirmation, PCa tissues were obtained from unstained slides with macrodissection. Total RNA was extracted from PCa tissues using superscript IV First-strand Synthesis system (Life Technology No. 18091200) and quantified using UV spectrophotometer NanoDrop 2000 (Thermo Scientific, USA). The cDNA templates were synthesized from RNA samples by SuperScript using random hexamers. RNA samples from the 404 patient samples were analyzed by qPCR to examine the expression of miR-1207-3p.
Gene expression was determined using power SYBR Green Real-Time PCR Master Mix (Life Technology No. 4367649) and 2.0 μl of cDNA template. qPCR was performed on an ABI7900 machine using the following amplification conditions: 10 minutes at 95°C followed by 45 cycles of 15 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C. All assays were carried out in triplicate to control for technical variance. Cycle threshold values were determined using the SDS ver2.3 software (Bio-Rad). MicroRNA 1207-3p expression was normalized with U6 expression within each sample. Relative quantification of target gene expression was evaluated using the comparative cycle threshold method.
Gene expression was determined using power SYBR Green Real-Time PCR Master Mix (Life Technology No. 4367649) and 2.0 μl of cDNA template. qPCR was performed on an ABI7900 machine using the following amplification conditions: 10 minutes at 95°C followed by 45 cycles of 15 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C. All assays were carried out in triplicate to control for technical variance. Cycle threshold values were determined using the SDS ver2.3 software (Bio-Rad). MicroRNA 1207-3p expression was normalized with U6 expression within each sample. Relative quantification of target gene expression was evaluated using the comparative cycle threshold method.
3
Ischemic Hemisphere Transcriptome Analysis
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Ischaemic cerebral hemisphere was removed from deeply anaesthetised rats at 24 h after reperfusion. Total RNA was extracted by using Trizol (Invirtogen) and reverse transcribed into cDNA with a Superscript First-Strand Synthesis System (Life Technologies, Grand Island, NY). Quantitative analysis was performed by using SYBR Green real-time PCR master mix (Life Technologies). GAPDH was used as the internal control in this study. The PCR primer sequences are listed in Table 1 .
4
Cinobufagin Inhibits Fibrosis Progression
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Cinobufagin (CBG) was purchased from Pusi Biotechnology Co. Ltd, and the purity of CBG was 99.21%. For each experiment, CBG was freshly prepared by dissolving in DMSO (Sigma, USA). Bleomycin (BLM) was acquired from Nippon Kayaku (Tokyo, Japan). TRIzol reagent was from Ambion Life Technology. DEPC TREATED H2O, RNASE AWAY H2O and SYBR Green Real-time PCR Master Mix were from Life Technologies. M-MLV Reverse Transcriptase was purchased from Promega, and PCR Buffer without MgCl2 and MgCl2 Stock Solution were acquired from Roche. In addition, Recombinant Ribonuclease Inhibitor, dATP, dTTP, dCTP, and dGTP were acquired from Takara. RIPA lysis buffer (middle) and the BCA kit were purchased from Beyotime Biotechnology. The primary antibodies described in the study include: anti-E-cadherin, anti-Vimentin, anti-fibronectin, and anti-collagen I (Affinity Biosciences, OH, USA) and anti-GAPDH, Smad3, P-Smad3, ERK, P-ERK, JNK, P-JNK, P38, P-P38 antibody (Cell Signaling Technology, Boston, United States); α-SMA and β-catenin antibody were from Santa Cruz Biotechnology (China) and Protein-Tech (China), respectively. The secondary antibodies anti-rabbit IgG (H+L) and anti-mouse IgG (H+L) were from Applygen (Beijing). Polyethylenimine, Linear purchased from Tianjin Hao Trading. β-catenin plasmid was purchased from MiaoLingPlasmid. ELISA kits were purchased from Beijing Suobao Technology.
5
Quantifying miRNA Expression in Prostate Cancer Cells
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The miRNeasy Mini kit (Qiagen, Germany) extracted total RNA from PC-3 and DU145 after transfection with each pre-miR precursors (miR-377 and miR-scrambled). Total RNA’s quantity and quality of total RNA were determined by Utilizing a UV spectrophotometric (IMPLEN, Munich, Germany) measure. The cDNA of the mRNA was generated using the cDNA synthesis kit (Fermentas, MA, USA). GAPDH (Table 1 ) was used as the endogenous control during the real-time PCR process. The SYBR Green real-time PCR Master Mix (Life Technology, MA, USA) 7.5 μL, 1 μL of cDNA, 0.5 mL of Forward Primer (10 pmol), 0.5 μL of Reverse Primer (10 pmol), and 5.5 μL of RNase-free H2O were combined to create the PCR mixes in 15 μL volumes. The statistical analysis for relative mRNA expression was conducted using the Relative Expression Software Tool (REST), as proposed by Pfaffl.
6
RNA Extraction and RT-qPCR Workflow
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The 1 ml aliquot of cells was centrifuged and resuspended in 350 μl of Buffer RLT (Qiagen). Samples were homogenised with a QIAshredder (Qiagen) and purified RNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions with on column Qiagen DNaseI digestion (Qiagen). Extracted purified RNA was resuspended in Nuclease-Free water (Promega) and concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific).
The purified RNA was converted into First-Strand cDNA using SuperScript II Reverse Transcriptase (Invitrogen) according to manufacturer’s instructions. The cDNA was then used for Real-Time PCR using the SYBR Green Real-Time PCR Master Mix (Life Technologies). Primer design was completed using Primer3 with the following conditions: primer size – 20 bp, primer melting point - 60 °C, primer G-C content – 55 % and product size – 100 bp minimum, 150 bp optimum, 200 bp maximum. The cycle conditions used were holding stage step 1–95 °C for 5 min, cycling stage step 1–95 °C for 10 s, step 2–60 °C for 20 s, step 3–72 °C for 20 s for a total of 40 cycles and a default melt curve stage.
The purified RNA was converted into First-Strand cDNA using SuperScript II Reverse Transcriptase (Invitrogen) according to manufacturer’s instructions. The cDNA was then used for Real-Time PCR using the SYBR Green Real-Time PCR Master Mix (Life Technologies). Primer design was completed using Primer3 with the following conditions: primer size – 20 bp, primer melting point - 60 °C, primer G-C content – 55 % and product size – 100 bp minimum, 150 bp optimum, 200 bp maximum. The cycle conditions used were holding stage step 1–95 °C for 5 min, cycling stage step 1–95 °C for 10 s, step 2–60 °C for 20 s, step 3–72 °C for 20 s for a total of 40 cycles and a default melt curve stage.
7
Real-time PCR for Gene Expression Analysis
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Real-time PCR was performed using the Roche 480 lightcycler and Sybr Green Real Time PCR Master Mix (Life Technologies). The cycling parameters were: 95°C, 10 s; 52°C, 20 s; and 72°C, 30 s, for a maximum of 45 cycles. Controls without reverse transcription were included in each assay. PCR primers specific to each gene were: Slc25a1 and Acly (Bio-Rad Laboratories; Prime PCR assay); Acss2: 5′-AAGAGCGGCAGTGGAAG-3′ (sense), 5′-TTCGATCCAGCACGTTGTAG-3′ (antisense); Gapdh: 5′-ACCACAGTCCATGCCATCAC-3′ (sense), 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense). Expression levels were normalized against Gapdh levels and are expressed as percent of control.
8
Bacterial 16S rRNA Gene Quantification
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The 16S DNA sequences of the bacteria, which characterize the three treatments, were used to design forward and reverse primers using the Primer Express V3.0 software (Thermo Fisher Scientific Inc., Waltham, MA, USA). The primer sequences are reported in Table 1 .
A QuantStudio 12K Flex unit (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to perform Real-Time PCR with the following mix: 5 μL of Sybr Green Real-Time PCR Master Mix (Life Technologies, Carlsbad, CA, USA), 0.1 μL of forward primer, 0.1 μL of reverse primer, and 1.4 μL of nuclease-free water. One μL of the sample was analyzed for each well. The cycling program consisted of 10 min of pre-incubation at 95 °C, 50 cycles of 15 s at 95 °C, and 1 min at 60 °C.
A QuantStudio 12K Flex unit (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to perform Real-Time PCR with the following mix: 5 μL of Sybr Green Real-Time PCR Master Mix (Life Technologies, Carlsbad, CA, USA), 0.1 μL of forward primer, 0.1 μL of reverse primer, and 1.4 μL of nuclease-free water. One μL of the sample was analyzed for each well. The cycling program consisted of 10 min of pre-incubation at 95 °C, 50 cycles of 15 s at 95 °C, and 1 min at 60 °C.
9
Validating RNA-seq Samples via qPCR
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To validate in vivo and ex vivo RNA-seq runs, primers were designed for selected genes (listed in Additional file 1 ). Also, some putative virulence factors were validated in these two samples, as well as in additional in vivo samples from a previous experiment [27 (link)]. Briefly, snatch-farrowed colostrum-deprived piglets were intranasally inoculated with 5 × 106 CFU of Nagasaki. After 1, 2 and 3 days post infection (dpi), pigs were euthanized and lungs were recovered. Bronchoalveolar lavage was performed and bacteria were obtained for RNA purification as described above. Samples were retrotranscribed using SuperScript® VILO™ Master Mix (Life Technologies). qPCR were performed in triplicates in the 7500 Fast Real-Time PCR System with SYBR® Green Real-Time PCR Master Mix (Life Technologies). Each sample belonged to one animal at the specific time post infection. Expression of the selected genes was compared to plate culture. The results were analysed using the ΔΔCt method.
10
Quantifying APH1 Expression in Yeast
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WT cells were grown overnight in YPD, washed, and incubated for 3 h in inducing (MM-KCl) or suppressing (MM-KH2PO4) medium and then snap-frozen in liquid nitrogen. The cells were homogenized by bead beating in the presence of glass beads and TRIzol (Ambion), and RNA was extracted following the manufacturer’s instructions. cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega). APH1 transcript was quantified by quantitative PCR (qPCR) using the SYBR green real-time PCR master mix (Life Technologies) on a Rotorgene 6000 qPCR machine (Corbett Research). APH1 expression was normalized against the expression of actin (ACT1) as a housekeeping gene before final quantification using the 2−ΔΔCT calculation method.
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