Total RNA of about 1000 antennae, 150 mouthparts (a piece of the head capsule anterior of the antennae), 600 legs, 50 heads (without antennae but including mouthparts), and 20 remaining bodies of sex-separated adults was isolated using the ZR Tissue and Insect RNA Micro Prep Kit (Zymo Research, Irvine, CA, USA), following the manufacturer´s protocol. The library preparations for RNA-Seq were performed using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) and cDNA libraries were amplified and sequenced using the cBot and HiSeq2000 from Illumina (paired end; 2 × 100 bp). Biological triplicates of female and male antennae were sequenced. For the other tissues, one male and one female sample were used and one additional mouthpart sample was obtained from unsexed beetles. The number of biological replicates was chosen based on previous publications using similar approaches [128 (link), 170 , 171 (link)]. Each biological replicate came from independently prepared and processed tissue. No technical replicates were performed. For details, see Dippel et al. [89 (link)].
Zr tissue and insect rna micro prep kit
Manufactured by Zymo Research
Sourced in United States
The ZR Tissue and Insect RNA Micro Prep Kit is a laboratory product designed for the isolation of high-quality total RNA from small tissue samples and insect specimens. The kit utilizes a fast and efficient column-based purification method to extract RNA from a variety of sample types.
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Lab products found in correlation
2 protocols using zr tissue and insect rna micro prep kit
1
Transcriptome Analysis of Insect Tissues
2
Quantitative Analysis of Fluorescent Reporters
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Total RNA was isolated from heterozygous third-instar larvae using the ZR Tissue and Insect RNA MicroPrep kit (Zymo Research) and 1 μg of RNA was transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). Primer pairs MFS17/MFS18, P16/P17 and MFS19/MFS20 were designed to compare the expression levels of EGFP and DsRed to the AmCyan control. The iQ SYBR Green Supermix (Bio-Rad)
was used for qPCR with 100 ng cDNA in a CFX96 Touch Real-Time time PCR Detection System (Bio-Rad). All reactions were performed on three biological and technical replicates. AmCyan was used as reference gene, and the 2 -∆∆Ct method [76] was used with all samples relative to the DsRed expression in V220. One-way ANOVA (SigmaPlot v12.5) was used to compare the relative expression levels of target genes and means were separated using Duncan's method.
was used for qPCR with 100 ng cDNA in a CFX96 Touch Real-Time time PCR Detection System (Bio-Rad). All reactions were performed on three biological and technical replicates. AmCyan was used as reference gene, and the 2 -∆∆Ct method [76] was used with all samples relative to the DsRed expression in V220. One-way ANOVA (SigmaPlot v12.5) was used to compare the relative expression levels of target genes and means were separated using Duncan's method.
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