Total RNA was extracted from cells using the Blood/Cultured Cell Total RNA Mini Kit (FAVORGEN, Ping Tung, Taiwan), followed by reverse transcription using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. cDNA was amplified for 40 cycles in a Light Cycler 480 (Roche) using LightCycler 480 SYBR Green I Master reagent (Roche). Expression of survivin was normalized to that of GAPDH mRNA (internal standard) by the ΔΔCt method. The primer pairs were as follows (final concentration 0.5 μM): human survivin, 5′-GGACCACCGCATCTCTACAT-3′ and 5′-GC ACTTTCTTCGCAGTTTCC-3′; human GAPDH, 5′-GAAA GGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATG GGATTTC-3′. Three independent experiments were performed.
Lightcycler 480 sybr green 1 master reagent
Manufactured by Roche
Sourced in Switzerland, United States
The LightCycler 480 SYBR Green I Master reagent is a ready-to-use master mix designed for real-time PCR applications. It contains SYBR Green I dye, DNA polymerase, and necessary reaction components.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 system, by Roche (10 mentions)
Lightcycler 480, by Roche (9 mentions)
Quantitect reverse transcription kit, by Qiagen (6 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Rneasy plus mini kit, by Qiagen (5 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (4 mentions)
Lightcycler 480 2, by Roche (3 mentions)
Omniscript reverse transcriptase kit, by Qiagen (3 mentions)
Tripure rna isolation reagent, by Roche (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Revertra ace qpcr rt master mix, by Toyobo (2 mentions)
Maxwell as3000, by Promega (2 mentions)
Cfx384, by Bio-Rad (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
Transcriptor high fidelity cdna synthesis kit, by Roche (2 mentions)
Lightcycler 96 instrument, by Roche (2 mentions)
Bioanalysis, by Agilent Technologies (2 mentions)
Taqman hydrolysis probe primer sets, by Thermo Fisher Scientific (2 mentions)
Abi 7500, by Thermo Fisher Scientific (2 mentions)
Ecodry cdna synthesis kit, by Takara Bio (2 mentions)
40 protocols using lightcycler 480 sybr green 1 master reagent
1
Quantification of Survivin mRNA Expression
2
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 mRNA extraction from cells as well as cDNA synthesis was performed using MPLC RNA IsoI. Kit (Roche) according to the manufacturer’s instructions. For PCR, the following primer pairs were used:
Gene expression was quantified by qRT-PCR on a LightCycler 480 II using LightCycler® 480 SYBR green I master reagent (Roche). The quantitative PCR was performed in a 20 µL reaction solution containing 6 µL nuclease-free water, 2 µL cDNA templates, 10 µL Master Mix and 1 µL each of forward and reverse primer. The amplification conditions were initial denaturation at 95°C for 15 min followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s and elongation at 72°C for 20 s. The experiment was performed in triplicates. A qualitative PCR was also performed in order to confirm the presence of single and appropriate bands for each primer set. PCR data were analyzed using the ΔΔCT method as previously described (40 (link)).
Gene expression was quantified by qRT-PCR on a LightCycler 480 II using LightCycler® 480 SYBR green I master reagent (Roche). The quantitative PCR was performed in a 20 µL reaction solution containing 6 µL nuclease-free water, 2 µL cDNA templates, 10 µL Master Mix and 1 µL each of forward and reverse primer. The amplification conditions were initial denaturation at 95°C for 15 min followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s and elongation at 72°C for 20 s. The experiment was performed in triplicates. A qualitative PCR was also performed in order to confirm the presence of single and appropriate bands for each primer set. PCR data were analyzed using the ΔΔCT method as previously described (40 (link)).
3
Analyzing Gene Expression in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA from flash-frozen gonadal fat and liver were extracted using the simplyRNA Tissue kit on a Maxwell AS3000 (Promega). cDNA was generated using the Transcriptor First Strand cDNA Synthesis kit (Roche). qPCRs were performed on a LightCycler 480 (Roche) and CFX384 (BioRad) with LightCycler 480 SYBR Green I Master reagent (Roche). B2m was used as a housekeeping gene to correct for starting amounts of cDNA in both gonadal fat and liver. Primers were purchased from Integrated DNA Technologies as custom DNA oligos and sequences are provided in Supplementary Table S2 .
4
Quantitative Real-Time PCR Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative real-time PCR reactions were done with the same batches of total RNA as were used for microarray analysis. cDNA synthesis was done as described60 (link). qRT-PCR analysis was performed using a LightCycler 480 II and LightCycler 480 SYBR Green I Master reagent (both Roche Diagnostics) according to manufacturer’s instructions. Putative heparin target genes were randomly selected to be corroborated by qRT-PCR. Human Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization of sample material. Data analysis was done as described previously61 (link). For qRT-PCR primer sequences see Supplementary Table S6 .
5
Quantitative PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were sacrificed at day 7 post irradiation and total RNA of the tumors isolated by TRIzol (Invitrogen) was reverse transcripted into cDNA by Omniscript reverse transcriptase kit (Qiagen, Düsseldorf, NRW, Germany). The quantitative PCR performed by the LightCycler® 480 SYBR Green I Master reagent (Roche, Basel, Switzerland) was analyzed by CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The mRNA expression of each gene was normalized for the housekeeping gene (β-actin) and the fold change of gene expression in each group was determined by the difference (ΔΔCt value) compared to the control group. The primers of genes are shown in Table 1 .
6
RT-qPCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT-qPCR analysis was performed using a LightCycler 480 II and LightCycler 480 SYBR Green I Master reagent (both Roche Diagnostics) according to manufacturer’s instructions. cDNA synthesis was carried out as described [63 (link)]. For normalization of sample material, human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used. Data analysis was carried out as described in Regl et al. [64 (link)]. For RT-qPCR primer sequences, see Table S4 .
7
Total RNA Isolation and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from mouse tissues using TriPure RNA isolation reagent (Roche) according to the product manual. To analyze N2 worms at day 11 of adulthood a total of approximately 3,000 worms per condition, divided into three biological replicates, was recovered in M9 buffer from NGM plates and lysed in the TriPure RNA reagent. Each experiment was repeated twice. Total RNA was transcribed to cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN). Expression of selected genes was analyzed using the LightCycler480 system (Roche) and LightCycler 480 SYBR Green I Master reagent (Roche). For worms two housekeeping genes were used to normalize the expression data, actin (act-1) and peroxisomal membrane protein 3 (pmp-3); for mice, the β2-microglobulin (B2m) gene was used as housekeeping reference.
8
Profiling Pluripotency Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine mRNA expression of the transcription factors SOX2, KLF4, cMYC, OCT4 and Nanog, total RNA was isolated from stromal cells (passage 1, n = 3 for each source) cultured in different media using High Pure RNA isolation kit (Roche Diagnostics, Rotkreuz, Switzerland) according to manufacturer’s protocol. cDNA synthesis was done as described previously [35 (link)]. qRT-PCR analysis was performed using a LightCycler 480 II and LightCycler 480 SYBR Green I Master reagent (both Roche Diagnostics) according to manufacturer’s instructions. For normalization of sample material, human Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used. Data analysis was done as described [36 (link)], heat maps were done using ClustVis tool [37 (link)]. For qRT-PCR primer sequences are according to Lee et al. [38 (link)]. For detailed sequence information see also Additional file 3 .
9
Quantitative RT-PCR Analysis of Par3 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 cell lines using a Cultured Cell Total RNA Purification Mini Kit (FAVORGEN, Ping Tung, Taiwan) followed by reverse transcription using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan) according to manufacturer’s instructions. cDNA was amplified for 40 cycles in a LightCycler 480 instrument (Roche, Basel, Switzerland) using LightCycler 480 SYBR Green I Master reagent (Roche). The primer sets used for qPCR are: for Par3, 5′-CGCTTGGAACATGGAGATGG-3 and 5′-ATCTCTGGGCTCTGGGTACC-3, for GAPDH, 5′-GAAAGGTGAAGGTCGGAGTC-3 and 5′-GAAGATGGTGATGGGATTTC-3. mRNA levels of each gene were normalized to GAPDH mRNA as an internal standard. Expression levels were calculated by the comparative Ct method using GAPDH as the endogenous reference gene.
10
Quantitative RT-PCR Analysis of Plant Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from tissues of tea plant as well as Arabidopsis thaliana using an RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s protocols. For qRT-PCR, 1 μg of total RNA was used to synthesize first-strand cDNA with PrimeScript RT enzyme together with gDNA eraser (Takara, Kyoto, Japan). qRT-PCR was performed on a Roche LightCycler 480 (Roche Diagnostics, Rotkreuz, Switzerland) using LightCycler 480 SYBR Green I Master Reagent (Roche Diagnostics, Rotkreuz, Switzerland). The gene-specific primer pairs used are listed in Table S3 . The polypyrimidine tract-binding protein (CsPTB1) gene was used as an internal control [72 (link)]. The relative expression levels were calculated using the 2−ΔΔCt method [73 (link)].
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!