mESC-CMs were isolated using Trypsin and placed in Trizol (Life Technologies). RNA isolation was performed following the manufacturer’s instructions and cDNA was generated using the High-Capacity cDNA reverse transcription kit (Applied Biosystems). All quantitative PCR reactions were performed using the Sybr Select qPCR mix (Thermo Fisher) with indicated primers (Table S5 ). Gene expression levels were normalized to GAPDH.
Sybr select qpcr mix
Manufactured by Thermo Fisher Scientific
Sybr Select qPCR mix is a real-time PCR reagent designed for quantitative gene expression analysis. It contains SYBR Green I dye, DNA polymerase, and necessary buffers and reagents for efficient and sensitive real-time PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Nb100 125, by Novus Biologicals (1 mentions)
4 protocols using sybr select qpcr mix
1
Isolation and qPCR Analysis of mESC-CMs
2
Clonal Analysis of Cardiac Progenitor Fate
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA isolation was performed using either RNeasy Micro Kit (Cat# 74004, Qiagen) or ARCTURUS® PicoPure® RNA Isolation Kit following the manufacturer’s instructions, and cDNA was generated using the high-capacity cDNA reverse transcription kit (Applied Biosystems). qPCR reactions were performed using the Taqman (Applied Biosystems) or Sybr Select qPCR mix (Thermo Fisher) with indicated primers. Gene expression levels were normalized to Gapdh. For the clonal cell-fate analysis, single Isl1-Cre RFP+, Cxcr4−, and Isl1-Cre RFP+, Cxcr4+ cells were sorted at day 5.5 into 384-well plates and allowed to grow and differentiate for 7 days. Appearance of colonies was visually confirmed by microscopy. RNA was isolated from 24 wells with colonies from Cxcr4− and Cxcr4+ sorted cells, respectively. Ct values < 30 were considered positive. All samples were also analyzed for gapdh to exclude false-positive results.
3
Cardiac Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from mouse hearts and cultured cardiomyocytes using TRIzol, and cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCRs were performed using the SYBR Select qPCR Mix (Thermo Fisher Scientific) with indicated primers. Gene expression levels were normalized to Gapdh or Rpl32.
4
ChIP-qPCR Analysis of Per2 in Mouse Heart
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA isolation for ChIP-qPCR analysis was performed using the protocol by van den Boogaard et al. (74 (link)). Briefly, several hearts (four to five hearts per sample) were isolated from P7 WT C57BL/6 mice and fixed in 1% paraformaldehyde followed by quenching with glycine. The heart tissue was subsequently homogenized and lysed using 1% SDS lysis buffer and protease inhibitor cocktail (Roche). DNA was fragmented using 20 cycles of 30 s “on” and then 30 s “off” of 50% power sonication, and DNA shearing efficiency was confirmed by DNA electrophoresis. Samples were subsequently incubated with protein G magnetic beads for 1 hour. Samples (2.5%) were kept as input (reference sample). Per2 ChIP-grade antibody (Novus, NB100-125) was added, and samples were incubated at 4°C overnight. Protein G magnetic beads were incubated for 1 hour and then pulled down. Chromatin was eluted from the beads and then uncrosslinked by incubating overnight at 65°C. DNA was purified using phenol-chloroform following ribonuclease A (RNase A) and proteinase K treatment (Thermo Fisher Scientific). DNA concentrations were calculated using the DNA Qubit 4 Fluorometer (Thermo Fisher Scientific). qPCR was performed using SYBR Select qPCR Mix (Thermo Fisher Scientific) with specific primers. Fold enrichment of DNA fragments compared to the input samples was calculated.
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!