Total RNA was isolated using the TRIzol reagent (Invitrogen, USA), following the manufacturer’s protocol, and RNA purity was detected using a NanoDrop 2000 spectrometer (Thermo Fisher Scientific, USA). The quantitative real-time polymerase chain reaction (PCR) was performed using SuperReal PreMix Plus (Invitrogen) in a StepOnePlus Real-time PCR Detection System (Applied Biosystems, CA, USA). Relative gene expression was calculated using the 2-△△Ct method. Human GAPDH was used as an endogenous control for mRNA expression in the analysis.
Superreal premix plus
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperReal PreMix Plus is a reagent kit designed for real-time quantitative PCR (qPCR) experiments. It contains a premixed solution of necessary components, including a proprietary DNA polymerase, dNTPs, MgCl2, and buffer, optimized for accurate and reliable quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Steponeplus real time pcr detection system, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrometer, by Thermo Fisher Scientific (3 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Abi 7500 thermocycler, by Thermo Fisher Scientific (1 mentions)
Primer express 3, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Fastquant rt kit with gdnase, by Tiangen Biotech (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Script cdna synthesis kit, by Bio-Rad (1 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
12 protocols using superreal premix plus
1
Quantification of Gene Expression
2
Synovial Tissue Analysis of Osteoarthritis
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Eight synovial tissues were obtained from four patients with OA and four normal controls. The written informed consent for use of their samples from every participant were provided in the present study. Ethical approval for this study was granted by the ethics committee of People’s Hospital of Deyang City (2017–043).
Total RNA was isolated with the Trizol reagent (Invitrogen, USA). The qRT-PCR reactions were performed based on SuperReal PreMix Plus (Invitrogen, USA) in ABI 7300 Real-time PCR Detection System. With 2-ΔΔCt method, relative gene expression was determined. The human GAPDH and ACTB were used as endogenous controls for mRNA expression, and the human hsa-U6 was used as endogenous controls for miRNA expression in analysis.
Synovial tissues were lysed on ice with RIPA lysis buffer, and then the supernatants were collected by centrifugation at 12,000 rpm at 4 °C for 30 min. The protein concentrations were detected with BCA protein assay. The protein extracts were separated by 10% SDS-PAGE, transferred onto PVDF membranes, and probed using primary and then secondary antibodies. The primary antibodies were as follows: rabbit anti-GAPDH, TIMP3 Antibody, CTSS Antibody and TLR7 Antibody. The blots were visualized with ECL reagent.
Total RNA was isolated with the Trizol reagent (Invitrogen, USA). The qRT-PCR reactions were performed based on SuperReal PreMix Plus (Invitrogen, USA) in ABI 7300 Real-time PCR Detection System. With 2-ΔΔCt method, relative gene expression was determined. The human GAPDH and ACTB were used as endogenous controls for mRNA expression, and the human hsa-U6 was used as endogenous controls for miRNA expression in analysis.
Synovial tissues were lysed on ice with RIPA lysis buffer, and then the supernatants were collected by centrifugation at 12,000 rpm at 4 °C for 30 min. The protein concentrations were detected with BCA protein assay. The protein extracts were separated by 10% SDS-PAGE, transferred onto PVDF membranes, and probed using primary and then secondary antibodies. The primary antibodies were as follows: rabbit anti-GAPDH, TIMP3 Antibody, CTSS Antibody and TLR7 Antibody. The blots were visualized with ECL reagent.
3
Tissue Expression Analysis of AAA
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A total of 9 tissue samples (including 3 normal samples and 3 small AAA samples and 3 large AAA samples) were collected from Shanxi Provincial People’s Hospital. The Ethical Committee of Shanxi Provincial People’s Hospital approved this study and the respective patient provided informed consent in a written form. Total RNA was isolated using TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocol, and RNA purity was detected using a NanoDrop 2000 spectrometer (Thermo Fisher Scientific, MA, USA). The quantitative real-time polymerase chain reaction (PCR) was performed Based on SuperReal PreMix Plus (Invitrogen) in a StepOnePlus Real-time PCR Detection System (Applied Biosystems, CA, USA), with the following primers: GAPDH (forward: 5’-GGACCTGACCTGCCGTCTAG-3’, reverse: 5’-GTAGCCCAGGATGCCCTTGA-3), HPSE(forward: 5’-TGCTATCCGACACCTTTGC-3’, reverse: 5’-TTGCCTCATCACCACTTCTAT-3’), G0S2 (forward: 5’-CCTCTTCGGCGTGGTGCTC-3’, reverse: 5’-CTGCTGCTTGCCTTTCTCC–3’) synthesized by shanghai GENE ray Biotech. GAPDH was then handled as an internalreference. The relative expression was calculated using the2^(-△Ct) method. P values <0.05 showed statistical significance.
4
Exploring the ceRNA Network in BRCA Patients
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Ten pairs of breast cancer tissues and corresponding adjacent nontumor tissues from BRCA patients were obtained from the Department of Breast Disease, The First Affiliated Hospital of Jiaxing University. The study was approved by the ethics committee, and written informed consent was obtained from all patients.
In this ceRNA network, we randomly selected six circRNAs, miRNAs, and mRNAs, respectively, and verified the prediction results' reliability and validity in BRCA patients using qRT-PCR. Total RNA was isolated using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol, and RNA purity was detected by NanoDrop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Based on SuperReal PreMix Plus (Invitrogen, USA) in StepOnePlus Real-time PCR Detection System (Applied Biosystems, Foster City, CA, USA), the qRT-PCR reactions were performed. The relative gene expression was calculated by 2-△△Ct. The human β-actin and human U6 were used as endogenous controls for mRNA and miRNA expressions in analysis, respectively. The human GAPDH was used as endogenous controls for circRNA expression in the analysis.
In this ceRNA network, we randomly selected six circRNAs, miRNAs, and mRNAs, respectively, and verified the prediction results' reliability and validity in BRCA patients using qRT-PCR. Total RNA was isolated using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol, and RNA purity was detected by NanoDrop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Based on SuperReal PreMix Plus (Invitrogen, USA) in StepOnePlus Real-time PCR Detection System (Applied Biosystems, Foster City, CA, USA), the qRT-PCR reactions were performed. The relative gene expression was calculated by 2-△△Ct. The human β-actin and human U6 were used as endogenous controls for mRNA and miRNA expressions in analysis, respectively. The human GAPDH was used as endogenous controls for circRNA expression in the analysis.
5
Intestinal Obstruction Vascular RNA Analysis
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In total, 23 blood samples were obtained from 13 patients with vascular intestinal obstruction and 10 healthy control patients. The total ribonucleic acid (RNA) was isolated with the Trizol reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions. The qRT-PCR reactions were performed using the ABI 7300 Real-Time PCR Detection System with SuperReal PreMix Plus (Invitrogen, USA). Relative gene expression was analyzed using the 2−ΔΔCT method. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin beta (ACTB) were used as endogenous controls.
6
Quantifying LUAD Gene Expression
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Thirteen samples were collected from 7 patients with non-metastatic LUAD and 6 patients with metastatic LUAD. We obtained written informed consent from every participant. The present study was approved by the Medical Ethics Committee of Meishan Cancer Hospital (201,908) and in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Total RNA was isolated with Trizol reagent (Invitrogen, USA). The qRT-PCR reactions were performed based on SuperReal PreMix Plus (Invitrogen, USA) in an ABI 7500 Real-time PCR Detection System. With the 2-ΔΔCt method, relative gene expression was determined. Human GAPDH and ACTB were treated as endogenous controls in the analysis.
7
Breast Cancer ceRNA Network Analysis
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Ten pairs of breast cancer tissues and corresponding adjacent non-tumor tissues from BRCA patients were obtained from Department of Breast Disease, The First A liated Hospital of Jiaxing University. The study was approved by the ethics committee and written informed consent was obtained from all patients. In this ceRNA network, we randomly selected six circRNAs, miRNAs and mRNAs respectively, and veri ed the reliability and validity of the prediction results in BRCA patients using qRT-PCR. Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol, and RNA purity was detected by NanoDrop 2000 spectrometer (Thermo Fisher Scienti c, Waltham, MA, USA). Based on SuperReal PreMix Plus (Invitrogen, USA) in StepOneplus Real-time PCR Detection System (Applied Biosystems, Foster City, CA, USA), the qRT-PCR reactions were performed. The relative gene expression was calculated by 2 -△△Ct . The humanβ-actin and human U6 was used as endogenous controls for mRNA and miRNA expression in analysis, respectively. The human GAPDH was used as endogenous controls for circRNA expression in analysis.
8
Quantification of C. elegans Gene Expression
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The total RNA of washed C. elegans (approximately 3000) was extracted through RNAeasy™ Plus Animal RNA Isolation Kit, and cDNA was synthesized by a FastQuant RT Kit (with gDNase). Moreover, qRT-PCR was performed with SuperReal PreMix Plus (SYBR Green), using the QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression levels were normalized to the housekeeping gene β-actin with the comparative 2−ΔΔCt (Ct, cycle threshold) method. The designed primer sequences are listed in Supplementary Table S1 .
9
RT-qPCR Gene Expression Analysis Protocol
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cDNA was synthesized from 1 μg of total RNA using a FastQuant RT Kit according to the manufacturer’s instructions at 4°C for 3 min, 42°C for 15 min, and 95°C for 3 min. The RT-qPCR was performed using a SuperRealPreMix Plus (SYBR Green) kit, a reaction volume of 20 μl (10 μl of PCR buffer, 0.6 μl of each primer [10 μM/μl], 3 μl of template cDNA, and 5.4 μl of DEPC H2O and 0.4 μl 50× ROX Reference Dye) and ABI-7500 thermocycler (Applied Biosystems). The thermocyling program was 94°C for 15 min, followed by 40 cycles of 95°C for 10 s and 60°C for 32 s. Fluorescence was measured at the end of every 60°C extension phase. Beta-actin of SBPHs or ecdysoneless (ECD) of Sf9 cells were used for normalization as housekeeping genes in respective experiments. RT-qPCR data were analysed using the Livak method (2−ΔΔCt) [63 (link)]. The experiments were repeated 3 times independently.
10
Quantitative PCR Analysis of P2X2 Receptor
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PBS was used to isolate and immediately wash the rat spinal cord dorsal horn lumbosacral enlargement. A FastQuant RT Kit with gDNase (Tiangen Biotech Co., Beijing, China) was used to synthesize cDNA with total RNA of 2 µg. TRNzol Universal Reagent (Beijing Tiangen) was used to prepare the total RNA samples. Primer Express 3.0 (Applied Biosystems, Inc., Foster City, CA, USA) was used to design the primers. The sequences were defined in the following way: P2X2 receptor, forward 5′-GGTGGTAGTGCCGTTTATCT-3′, reverse 5′-AAGGGCGGTGTCATTGGA-3′; β-actin, forward 5′-AAGATCCTGACCGAGCGTGG-3′, reverse 5′-CAGCACTGTGTTGGCATAGAGG-3′. The ΔΔCT method was used to quantify the gene expression, with the threshold cycle denoted by CT. The relative levels of the target genes normalized to those of the samples with the lowest CT were reported as 2−ΔΔCT. A SuperReal PreMix Plus (SYBR Green) was used to perform quantitative PCR in an ABI PRISM® 7500 Sequence Detection System (Applied Bio-systems, Inc.).
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