mtDNA copy number quantification was conducted on tissues that were derived from the same tissue collection group (but not identical mice) as the ones used for RNA-seq. To analyze mtDNA copy number, total DNA of WAT samples was isolated using the DNA Blood and Tissue kit (69506, Qiagen) with an additional centrifugation step at 200g for 5 min after lysis in ATL buffer. DNA concentrations were quantified using the Qubit dsDNA BR Assay kit (Q32853, ThermoFisher Scientific). QPCR was carried out in a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) using the Taqman Universal PCR Master Mix (Applied Biosystems). Reactions were run in quadruplicates on 384-well plates using 5 ng of total DNA per reaction. Specific Taqman probes were used to quantify the nuclear 18S gene, (Hs99999901_s1) and the Rnr2 (Mm04260181_s1), Atp6 (Mm03649417_g1) and Cox1 (Mm04225243_g1) genes for mtDNA (ThermoFisher Scientific). Data were analyzed using a standard curve method and relative mtDNA content was calculated by the ratio of mtDNA probes relative to genomic DNA (mtDNA/18S). Results were normalized to the relative mtDNA content of the AL control group.
Specific taqman probes were used
Manufactured by Thermo Fisher Scientific
Taqman probes are a type of fluorescent oligonucleotide probe used in real-time PCR (polymerase chain reaction) assays. They are designed to hybridize to a specific DNA sequence within the amplified region, allowing for the detection and quantification of the target DNA during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using specific taqman probes were used
1
Quantification of Mitochondrial DNA Content
2
Quantification of Mitochondrial DNA Content
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mtDNA copy number quantification was conducted on tissues that were derived from the same tissue collection group (but not identical mice) as the ones used for RNA-seq. To analyze mtDNA copy number, total DNA of WAT samples was isolated using the DNA Blood and Tissue kit (69506, Qiagen) with an additional centrifugation step at 200g for 5 min after lysis in ATL buffer. DNA concentrations were quantified using the Qubit dsDNA BR Assay kit (Q32853, ThermoFisher Scientific). QPCR was carried out in a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) using the Taqman Universal PCR Master Mix (Applied Biosystems). Reactions were run in quadruplicates on 384-well plates using 5 ng of total DNA per reaction. Specific Taqman probes were used to quantify the nuclear 18S gene, (Hs99999901_s1) and the Rnr2 (Mm04260181_s1), Atp6 (Mm03649417_g1) and Cox1 (Mm04225243_g1) genes for mtDNA (ThermoFisher Scientific). Data were analyzed using a standard curve method and relative mtDNA content was calculated by the ratio of mtDNA probes relative to genomic DNA (mtDNA/18S). Results were normalized to the relative mtDNA content of the AL control group.
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!