Computational protocol: Protruding Structures on Caterpillars Are Controlled by Ectopic Wnt1 Expression

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Protocol publication

[…] A short interfering RNA (siRNA) for Wnt1 targeting the sequence 5′-AAGAAGATTGGCCAGAGAGAA- 3′ was designed using siDirect version 2.0 (http://sidirect2.rnai.jp) according to the criterion [, ] (purchased from FASMAC Co., Japan). For negative controls, the Universal Negative Control siRNA (Nippon Gene Co., Japan) was used. siRNAs (0.5 μl; 250 μM) were injected into the hemolymph of 3rd instar larvae with glass needles using a microinjector. To introduce siRNA into the region only around one side of the knob [], soon after the injection, PBS droplets were placed near the injection site and knobbed region in the 5th larval segment and electrical stimuli applied as described above (.). [...] cDNAs were synthesized from the larval epidermis of B. mori in the 4th larval stage and B. alcinous in the 4th and 5th larval stages. After anaesthetization of the larvae on ice, the epidermis was dissected in cold PBS. When dissecting the epidermis, only the dorsal part was used, subcutaneous tissues such as muscle and fat body were trimmed away carefully with forceps. Total RNA was extracted using TRI reagent (Sigma) and purified with standard phenol/chloroform extraction following treatment with DNase I (TaKaRa) for 15 min at 37°C. The RNA was quantified by spectrophotometry (NanoDrop, Thermo Scientific) and the A 260/A 280 ratio confirmed to be 1.8–2.0. Total RNA (0.1–1 μg) was used in the reverse transcriptase reactions, which were performed per the manufacturer’s instructions using random hexamers and M-MuLV reverse transcriptase (GE Healthcare; First-Strand cDNA Synthesis Kit).Quantitative RT-PCR was performed using the StepOne system with the Power SYBR Green PCR master mix (Applied Biosystems) using a MicroAmp Fast Optical 48-well reaction plate (Wako, Japan) under the manufacture’s recommended condition (denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s). Relative gene expression values were estimated using relative standard curve method. The primers for B. alcinous for Wnt1 were qBa_wnt1_01 (5′-GATTCCGATTCAGCCGGGAGTTCGT-3′, 5′-ACATGCGCTCTGCCGGCTTCGTT-3′) and qBa_rpL3_01 (5′-GACCGTATGGGCAGAACATATGTCTG-3′, 5′-TCTTGCTTGACTTAG TAAAGGCCTTCTTC-3′) for ribosomal protein L3 (rpL3) used as an internal control [, , ] (). All primers were designed to target products < 120 bp using Primer Express 2.0 software (Applied Biosystems). Standard curves were generated for all primer pairs to estimate efficiency and to confirm the limit of detection. The specificity of primer pairs was confirmed by melting curves.In B. mori, semi-quantitative RT-PCR was performed using the following primers: cWnt1_02-F (5′-GGCGGTTCACGCTACGCTA-3′) and cWnt1_05-R (5′-ATCCACAATTTTTCCGAACAAGTT-3′) for Wnt1, and rpL3-5 (5′-AGCACCCCGTCATGGGTCTA-3′) and rpL3-3 (5′-TGCGTCCAAGCTCATCCTGC-3′) for an rpL3 internal control [, ]. The amplification program was as follows: 95°C for 2 min, then 35 cycles of 95°C for 15 s, 55°C for 30 s, 72°C for 30 s and final extension 72°C for 4 min. […]

Pipeline specifications

Software tools siDirect, Primer Express
Applications qPCR, Non-coding RNA analysis
Organisms Caenorhabditis elegans, Atrophaneura alcinous, Bombyx mori, Drosophila melanogaster