Total RNAs were isolated from patient samples and human cells with Trizol reagent, and quantified using a NanoDrop spectrophotometer (Thermo Scientific, Rockford, IL). Regular and stem-loop reverse transcription of mature miR-1291 were conducted as described previously [30 (link), 60 (link)]. RT-qPCR was performed on a MyIQ real-time PCR system (Bio-Rad, Hercules, CA). The cycle number (CT) at which the amplicon concentration crossed a defined threshold was determined for each individual miRNA. The relative level of each analyte over internal standard (glyceraldehyde-3-phosphate dehydrogenase, or U6) was calculated as 2−ΔΔCT , where ΔΔCT = ΔCT treatment group (analyte – internal standard) – ΔCT control group (analyte – internal standard).
Myiq real time pcr system
Manufactured by Bio-Rad
Sourced in United States, Germany
The MyiQ Real-Time PCR System is a laboratory instrument used for conducting real-time polymerase chain reaction (PCR) experiments. It is capable of precisely measuring and analyzing the amplification of DNA sequences in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Iscript cdna synthesis kit, by Bio-Rad (10 mentions)
Iq sybr green supermix, by Bio-Rad (9 mentions)
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Rneasy kit, by Qiagen (5 mentions)
Maxima sybr green fluorescein qpcr master mix, by Thermo Fisher Scientific (4 mentions)
Iq5 program, by Bio-Rad (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Transcriptor first strand cdna synthesis kit for rt pcr, by Roche (2 mentions)
Sybr green dynamo hs kit, by New England Biolabs (2 mentions)
Superscript 3 system, by Thermo Fisher Scientific (2 mentions)
Isolate 2 rna mini kit, by Meridian Bioscience (2 mentions)
Iq syber green supermix, by Bio-Rad (2 mentions)
Sybr green, by Bio-Rad (2 mentions)
Transcriptor first strand cdna synthesis kit for rt pcr, by Thermo Fisher Scientific (2 mentions)
Iq5 gene expression analysis, by Bio-Rad (2 mentions)
59 protocols using myiq real time pcr system
1
Quantitative Analysis of miRNA Expression
2
Analyzing Gene Expression in Breast Cancer Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from MCF-7 and MDA-MB-231 cells, respectively, using the RNeasy MinElute Cleanup Kit (Qiagen GmbH, Hilden, Germany). cDNA was synthesized with SuperScript III Reverse Transcriptase (Life Technologies) and random hexamer primers (200 ng; #SO142, Fisher Scientific GmbH, Schwerte, Germany). SYBR Green real time PCR was carried out with 13 μl SYBR Green Master mix (ABsolute QPCR SYBR Green Mix, Fisher Scientific), 10 nM primer and 15 ng cDNA in a final reaction volume of 20 μl. All samples were run in duplicate. RPL27 was used as a reference gene. The characteristics of the primers are shown in Table S1 .
Thermal cycling was performed on a MyiQ™ real-time PCR system (Bio-Rad Laboratories GmbH, Munich, Germany) with the following protocol: 95 °C for 10 min, 45 cycles at 95 °C for 30 sec → 60 °C for 30 sec → 70 °C for 30 sec, and 72 °C for 5 min. To check for specificity of the primers the temperature was ramped up from 55 °C to 95 °C (at the rate of 0.5 °C/s). Melting curve analysis was performed according to the dissociation stage data and reactions with a single peak at the expected Tm were considered for further analysis. Calculation of the gene expression level was carried out with the program Rest 2005 (Corbett Research, Sydney, Australia and Technical University of Munich, Germany) using the comparative threshold method 2−ΔΔCT.
Thermal cycling was performed on a MyiQ™ real-time PCR system (Bio-Rad Laboratories GmbH, Munich, Germany) with the following protocol: 95 °C for 10 min, 45 cycles at 95 °C for 30 sec → 60 °C for 30 sec → 70 °C for 30 sec, and 72 °C for 5 min. To check for specificity of the primers the temperature was ramped up from 55 °C to 95 °C (at the rate of 0.5 °C/s). Melting curve analysis was performed according to the dissociation stage data and reactions with a single peak at the expected Tm were considered for further analysis. Calculation of the gene expression level was carried out with the program Rest 2005 (Corbett Research, Sydney, Australia and Technical University of Munich, Germany) using the comparative threshold method 2−ΔΔCT.
3
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from bone, bone marrow, and cell cultures using TriReagent (Sigma-Aldrich) and reverse transcribed using Superscript II (Invitrogen) and random hexamers. cDNA was combined with 0.5 μM each of the forward and reverse primers (Supplemental Table S1) and iQ SYBR Green Supermix and run in the MyIQ Real-Time PCR system (Bio-Rad, Hercules, CA). Raw data were analyzed with PCR Miner (Zhao and Fernald, 2005 (link)) and normalized using the internal control transcript for ribosomal protein L32.
4
Quantification of Mouse Prostanoid Receptors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 μg of total RNA, extracted with TRI Reagent (Ambion, Life Technologies), was reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit for RT-PCR following the indications of the manufacturer (Roche). Real-time PCR was conducted with SYBR Green (Roche) on a MyiQ Real-Time PCR System (Bio-Rad). The TaqMan probes for mouse EP1, EP2, EP3, EP4, P2Y2, and P2Y4 used in this study were purchased from Applied Biosystems and experiments for validation of amplification efficiency were performed for each TaqMan probe set [28 (link), 29 (link)]. PCR thermocycling parameters were 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Each sample was run in duplicate and was normalized with the expression of 36B4. The fold induction (FI) was determined in a ΔΔCt based fold-change calculations (relative quantity, RQ, is 2−ΔΔCt).
5
RNA Extraction and qPCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with ice-cold sterile PBS. Trizol® Ambion (Madrid, Spain) protocol was applied for RNA extraction (1 mL/plate sample) [28 (link)]. RNA was quantified by ND-1000 (Nanodrop Technologies; Madrid, Spain) and the RNA integrity number (RIN) was calculated. RNA was retrotranscripted to cDNA with Transcriptor First-Strand DNA cDNA Synthesis (Roche) following the manufacturer’s instructions. qPCR assays were carried out with 5 µL cDNA + 10 µL SYBR® Green PCR Master Mix cocktail (Applied Biosystems) + 250 nM forward and reverse primers (Table 2 ). RLPLP0 was chosen as a house-keeping endogenous control for normalization purposes. Three RNA pools of four donors each were evaluated for each condition, by triplicate. qPCR reaction was carried out in MyIQ RealTime PCR System (BioRad). Result analysis was conducted with IQ5 program (BioRad), following the ΔΔCt method.
6
RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA isolation and real-time polymerase chain reaction (PCR) were performed as described previously (Zhang et al., 2019b (link)). Total RNA was prepared using the Isolate II RNA Mini Kit (Bioline USA, Inc.), and cDNA was then synthesized with the iScript cDNA Synthesis Kit (Bio-Rad). Subsequently, quantification of gene expression was performed in duplicate using iQ SYBR Green Supermix (Bio-Rad) with detection on a MyiQ Real-Time PCR System (Bio-Rad). The reaction cycles used were 95°C for 5 minutes, 40 cycles at 95°C for 15 seconds, and 58°C for 1 minute, and this was followed by melt curve analysis. Relative gene expression quantification was based on the comparative threshold cycle method (∆∆Ct) with normalization of the raw data to the housekeeping gene (18S ribosomal RNA).
7
RT-qPCR Gene Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RT-qPCR analysis, we synthesized first-strand cDNAs from 5 μg total RNA using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) using the provided protocol. A 1:40 dilution of the cDNAs was used for RT-qPCR. The amplification parameters were as follows: 15 min of denaturation and enzyme activation at 95 °C; followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 30 s; with a final step performed at 65–95 °C (1 °C/s) for melting curve analysis. The amplified signals were detected using a MyiQ real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Premix Ex TaqTM (TOPrealTM qPCR 2X PreMix, www.enzynomics.com , Yuseong-gu, Daejeon, Korea). The data were normalized based on the expression of rice UBIQUITIN5, and the relative gene expression was analyzed using the 2−ΔΔCt method or the 2−ΔCt method. Primer sequences used for RT-qPCR analysis are listed in Table S2 .
8
Liver Tissue Gene and Protein Expression Analysis
9
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the spinal cord or from primary cell cultures using Trizol (Invitrogen, CA, USA). First-strand cDNAs were synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Fermentas, Vilnius, Lithuania). qPCR was performed on a MyiQ Real Time PCR system (Bio-Rad, Hercules, CA, USA). Gene expression was expressed as the mRNA level, which was normalized to the mRNA level of a standard housekeeping gene (Gapdh) using the ΔΔCT method. At least three independent experiments were performed for each set of PCR analyses. The primers used in this study are listed in Supplementary Table 1 .
10
RT-qPCR Gene Expression Analysis
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!