Measuring PINK1 mRNA Expression in Drosophila

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.

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Thomas R.E., Andrews L.A., Burman J.L., Lin W.Y, & Pallanck L.J. (2014). PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix. PLoS Genetics, 10(5), e1004279.

Publication 2014

TRIzol iScript cDNA Synthesis kit Brilliant III Ultra-Fast SYBR Green QPCR Master Mix Opticon 2 machine

Cdna Expression gene Fast green Genotypes Mitochondrial Mrna Primers Reverse transcription Synthesis Trizol

Corresponding Organization :

Other organizations : University of Washington

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Variable analysis

independent variables

Genotype (elav-GAL4; Control-R/+ and elav-GAL4; Control-R/PINK1-myc)

dependent variables

PINK1 mRNA expression
Mitochondrial DNA abundance
Nuclear DNA abundance

control variables

Rap2l gene expression (used as internal control)
Reverse transcription and qPCR conditions (same for all samples)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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