Drosophila RNA Extraction and qRT-PCR

5 adult flies per biological replicate were homogenised in TRIzol and RNA extraction was carried out using Direct-zol RNA MiniPrep kit (Zymo Research, Z2050). DNA was removed with Turbo DNase free (Ambion, AM1907), and cDNA synthesis was performed using a Maxima First Strand kit (ThermoFisher, K1641) followed by a qRT-PCR using SYBR green (ThermoFisher, 4,309,155) with a CFX96 Touch Real-Time PCR Detection System. The relative transcript levels of each target gene were normalised against RpL32 and Tubulin (Tub84B) mRNA levels; quantification was performed using the comparative C_T method [37 (link)]. Primers used:

Tubulin - Fwd: TGGGCCCGTCTGGACCACAA; Rev: TCGCCGTCACCGGAGTCCAT [38 (link)],

RPL32 - Fwd: GCCGCTTCAAGGGACAGTATCTG; Rev: AAACGCGGTTCTGCATGAG [39 (link)],

Pink1 - Fwd: AACAGTCCGGAGATCCTACAG; Rev: GACGACCCTCGCACATAA [40 (link)], and.

Parkin - Fwd: AGTACACCGTGGACCCAAAT; Rev: TGTGCTGACTTTGATGGTGA [40 (link)].

Free full text: Click here

Martinez A., Sanchez-Martinez A., Pickering J.T., Twyning M.J., Terriente-Felix A., Chen P.L., Chen C.H, & Whitworth A.J. (2024). Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models. Molecular Neurodegeneration, 19, 12.

Publication 2024

Direct-zol RNA MiniPrep kit Turbo DNase free Maxima First Strand kit SYBR green CFX96 Touch Real-Time PCR Detection System

Corresponding Organization : University of Cambridge

Other organizations : National Health Research Institutes

Top 5 similar protocols

Variable analysis

independent variables

None explicitly mentioned

dependent variables

Relative transcript levels of Pink1 and Parkin genes

control variables

Number of adult flies per biological replicate (5)
RNA extraction method (Direct-zol RNA MiniPrep kit)
DNA removal method (Turbo DNase free)
CDNA synthesis method (Maxima First Strand kit)
QRT-PCR method (SYBR green, CFX96 Touch Real-Time PCR Detection System)
Normalization reference genes (RpL32 and Tub84B)

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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?

Revolutionizing how scientists
search and build protocols!