Rnagents total rna isolation kit

Nematodes were cultured on Escherichia coli strain OP50 on NGM agar plates [20] (link). RNA was extracted from 580 mg (for a culture grown at 20°C, called PDT) and 730 mg (for a culture grown at 20°C and subsequently brought down to 4°C, called PDF) of P. davidi CB1 (wet weights), respectively. Total RNA (930 µg for PDT and 750 µg for PDF) was prepared with RNagents total RNA Isolation Kit (Promega). Poly(A)+ RNA (2.9 µg for PDT and 5.5 µg for PDF) were isolated with Illustra mRNA Purification Kit (GE healthcare). 2 µg of poly(A)+ RNA was then used to generate a cDNA library with Cloneminer cDNA library Construction Kit (GE healthcare).

The Papilio retina contains five opsin mRNAs each encoding Papilio xuthusultraviolet-absorbing (PxUV), PxB (blue), PxL1 (green), PxL2 (green) and PxL3 (red) (Kitamoto et al., 1998 (link), 2000 (link)). We determined the post-pupation day at which each of these opsin mRNAs became detectable by reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from the eye tissues of the pupa every 24 h using the RNAgents Total RNA Isolation Kit (Promega), and cDNA was synthesized using oligo-dT primer by reverse transcription. The primers were designed to amplify 200–300 bp fragment of one of the opsin mRNA. The sequences of the primers are as follows: PxUVF (PxUV-Forward), TGAAT TCACA TATAC TGACC CAACG CG; PxUVR (PxUV-Reverse) GGAAA GCTTT CCATT ATTCA CGCCA GTTC; PxBF, AGAAT TCTCC AACGA ACGAT GCAAT CG; PxBR, CGAAA GCTTT CGGAG TCCAT ACAAC AAGC; PxL1F, TGAAT TCAAC GACGA CGAAT GTTTG CG; PxL1R, TAAAA GCTTT TACTA TCGCA GGCTA AC; PxL2F, GGAAT TCCCC TAAGG ATCTG ATACT GC; PxL2R, ACCAA GCTTG GTACA CAGCT TGTTT CATC; PxL3F, TGAAT TCAAC CAACG ATGAC GACTT GG; PxL3R, GATAA GCTTA TCACA CGAGG ATAGT AGGG. We also used primer sets for amplifying cDNA of actin (forward = CAYAC NGTIC CNATH TAYGA RGG; reverse = TCIGC DATNC CNGGR TACAT NGT).

Genomic DNA from C. purpurea was isolated as described by Cenis [47 (link)]. For Southern blot analysis, 5–10 μg of digested genomic DNA were separated via gel electrophoresis in a 1 % agarose gel with salt-free buffer [47 (link)] and transferred to a nylon membrane (Nytran SPC; Whatman). For the isolation of RNA, the RNAgents total RNA isolation kit (Promega GmbH, Mannheim, Germany) was used. For northern blotting, 20 mg RNA were used for the separation on a 1 % (w/v) agarose gel containing 1 % (v/v) formaldehyde and afterwards transferred to a nylon membrane (Nytran SPC; Whatman). For southern as well as northern hybridization, 32P-labeled probes were generated using the random oligomer-primer method, and hybridized to the membranes. PCR reactions were performed using either the BioTherm Taq DNA Polymerase (GeneCraft, Germany) or the proof reading Phusion DNA polymerase (Finnzymes, Finland). Primers were synthesized by Biolegio (Nijmegen, Netherlands).