Quantitative RT-PCR Analysis of Homozygous Genes

Quantitative RT-PCR was performed on four biological with two technical replicates each, probing homozygous H^WA and H^NN versus control H^cwt. Poly(A⁺) RNA was prepared from 12 third-instar wandering larvae with the PolyATtract^® System 1000 (Promega, Walldorf, Germany), followed by a DNase I (RNase free) digest (New England Biolabs, Ipswich, MA, USA). cDNA was synthesized from around 250 ng mRNA using the qScriber cDNA Synthesis Kit (HighQu, via Biozol, Eching, Germany). Real-time qPCR was performed as described before using the Blue S’Green qPCR Kit (Biozym, Scientific GmbH, Hessisch Oldendorf, Germany) on around 5 ng of cDNA and the MIC magnetic induction cycler (bms, via Biozym Scientific GmbH, Hessisch Oldendorf, Germany), including target and no-template controls [40 (link)]. Internal reference genes were cyp33 and Tbp. Primer pair sequences are listed at the DRSC FlyPrimer bank [50 (link)]: cnc (PP60393), cyp33 (PP14577), ewg (PP35080), GstD1 (PP16044), mTFB2 (PP26980) and Tbp (PP1556). Respective oligonucleotides were obtained from Microsynth (Balgach, Switzerland). The micPCR^® software version 12.2 was used for relative quantification of the data, based on REST and taking target efficiency into account [51 (link)].

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Mönch T.C., Smylla T.K., Brändle F., Preiss A, & Nagel A.C. (2024). Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development. Genes, 15(5), 552.

Publication 2024

PolyATtract® System 1000 Blue S’Green qPCR Kit

Corresponding Organization : University of Hohenheim

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Variable analysis

independent variables

Genotype: homozygous H^WA and H^NN versus control H^cwt

dependent variables

Expression levels of the following genes: cnc, cyp33, ewg, GstD1, mTFB2, Tbp

control variables

Third-instar wandering larvae used as samples
Poly(A+) RNA prepared using PolyATtract® System 1000 (Promega)
DNase I (RNase free) digest (New England Biolabs)
CDNA synthesis using qScriber cDNA Synthesis Kit (HighQu)
Real-time qPCR using Blue S'Green qPCR Kit (Biozym) and MIC magnetic induction cycler (bms)
Internal reference genes: cyp33 and Tbp
Primer pair sequences obtained from DRSC FlyPrimer bank
Relative quantification using micPCR® software version 12.2, based on REST and taking target efficiency into account

controls

Target controls
No-template controls

Annotations

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