Total RNA was extracted from 20 female flies per condition using TRIzol reagent (Thermo Fisher, Waltham, USA), chloroform, and isopropanol. Genomic DNA was removed from the extracted RNA using Turbo DNA-free kit (catalog no. AM1907, Invitrogen, USA), and cDNA was generated using Superscript III (catalog no. 18080051; Invitrogen, USA) following the manufacturer’s instructions. qPCR was performed in Maxima SYBR Green master mix (catalog no. K0221; Thermo Fisher, USA), using 100 ng of cDNA as the template and 10 μM target gene-specific primers. qPCR was performed with CF96 Touch real-time PCR detection system (Bio-Rad). The primers used are presented in Table 1 . RpL32 transcript levels were used for normalization.
Cf96 touch real time pcr detection system
Manufactured by Bio-Rad
The CF96 Touch real-time PCR detection system is a laboratory equipment designed for quantitative polymerase chain reaction (qPCR) analysis. It is capable of detecting and quantifying targeted DNA or RNA sequences in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Maxima sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (1 mentions)
Sybr green pcr kit, by Bio-Rad (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
2 protocols using cf96 touch real time pcr detection system
1
Quantitative Gene Expression Analysis in Drosophila
2
Extracting RNA from Mouse Lung
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To extract RNA from mouse lung, tissue was lysed and homogenized with Buffer RLT Plus (supplied with the RNeasy® Plus Mini Kit; Qiagen). The lysate was passed through a genomic DNA eliminator spin column, ethanol was added, and the sample was applied to a RNeasy MinElute spin column according to the manufacturer’s instructions. An iScript™ Select cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) was used for reverse transcription using oligo (dT) primers and random hexamer primer mix. An SYBR green PCR kit (Bio-Rad) was used for quantitative real-time PCR and was performed and analyzed on a CF96 Touch™ Real-Time PCR Detection System (Bio-Rad). The sequences of the primer pairs are listed in electronic supplementary Table 1.
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