For measurement of PINK1 mRNA, total RNA was extracted from elav-GAL4; Control-R/+ and elav-GAL4; Control-R/PINK1-myc using TRIzol (#15596-026, Life Technologies). Reverse transcription was done using the iScript cDNA Synthesis kit (#170-8890, Bio-Rad) and diluted 1∶50 and 1∶300 before use in qPCR reactions. Primer sequences for PINK1 and Rap2l were obtained from the FlyPrimerBank [47] (link). Primer pairs PA60267 and PP23832 were used for PINK1, and primer pair PP8673 for Rap2l. The log2 method was used to calculate fold change. Rap2l was used as the internal control, as the expression of this gene has been reported as the most invariant across different genotypes and ages [48] (link). qPCR was performed using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (#600882, Agilent Technologies) and a Bio-Rad Opticon 2 machine. Mitochondrial and nuclear DNA abundance were measured by using the DNA extraction method and primers described in a published report [49] (link). qPCR of mitochondrial and nuclear DNA was performed as described above.
Opticon 2 machine
Manufactured by Bio-Rad
The Opticon 2 machine is a real-time PCR detection system designed for quantitative gene expression analysis. It features a 96-well sample capacity and utilizes a stable thermal block design to ensure accurate and reproducible results. The Opticon 2 machine supports a wide range of fluorescent dyes and probes, enabling flexible experimental setups.
Automatically generated - may contain errors
2 protocols using opticon 2 machine
1
Measuring PINK1 mRNA Expression in Drosophila
2
Analyzing IFN-α/β and Cytokine Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
cDNA was prepared from purified RNA by reverse transcribing 1.0 µg of each RNA sample using a SABiosciences first-strand kit (C-03; SABiosciences, Frederick, MD). The 20-µl final volume of cDNA was diluted with 1,275 µl of water and added to 1,275 µl of 2× SABiosciences RT2 SYBR green master mix (PA-010). A 25-µl portion of this mixture was dispensed into each well of a SABiosciences RT2 profile IFN-α/β signaling (PAMM-14) or cytokine (PAMM-11) PCR array. RT-PCR was carried out on a Bio-Rad Opticon2 machine (Bio-Rad, Hercules, CA). Statistical analysis was performed using GraphPad software (Instat).
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!