Nucleic Acids Research, 2011, Vol. 39, No. 7 2703 Table 1. List of primers and oligonucleotides used in experiments described in this article RDmL22likeBamHI_shortened 50-GTCACGGATCCTTAGGCAAAGGTTTTTCCGCCATTG-30 Name FDmL22likeBamHI RDmL22likeBamHI FL22like_exon1/2 RL22like_novel exon2/3 RDmL22-likeFLAG-BamH1 FpEXP5-T7 FL22likeFull RL22likeFull FL22likeShort RL22likeShort FBeta2Tubulin RBeta2Tubulin FVasa RVasa FrpL32 RrpL32 FL22-like_startcodon FL22 RL22 RL22like_exon1 RL22like_exon3 RL22like full speciﬁc Sequence 50-GTCACGGATCCATGAGTTCCCAGACGCAGAAAAAGAATGCTTCCAA-30 50-GTCACGGATCCTTAGGCAAAGGTTTTTCCGCCATTGTCGTCGGCAA-30 50-GATACTAATTTCTTTGGAGAATG-30 50-TTAGGCAAAGGTTTTTCCGCCATTGTCGTCGGCAAGAGG-30 50-GTCACGGATCCTTACTTGTCATCGTCATCCTTGTAGTCGCCGCGGCCGAT GGCAAAGGTTTTTCCGCCATTGTCGTCGGCAA-30 50-CCGCGAAATTAATACGACTCACTATAGGGAGACACACGACGG-30 50-GCTCAACCAGCTGAAGGATCA-30 50-GAAGTAGCGCTTGGAGAAGTGC-30 50-CAGAATGCTTCACGCAAGAACTT-30 50-AATGTCGTCGGCAAGAAGC-30 50-GTGCTGAACTGGTGGATTCCGT-30 50-GGTCAGCTGGAAGCCCTGAA-30 50-CTGTACGAAAACGAGGATGGTGA-30 50-ACCACCGTCCCCTCTTTCA-30 50-CTAAGCTGTCGCACAAATGG-30 50-ACGCACTCTGTTGTCGATACC-30 50-ATGAGTTCCCAGACGCAGAAAAAGAATGCTTCCAAGGCC-30 50-AAGATGGCTCCTACCGCCAAGACCAACAAGGG-30 50-TTACTCGGCATCGTCGTCCTCATCG-30 50-GGAAGCATTCTTTTTCTGCGTCTGGGAACTC-30 50-TTAGGCAAAGGTTTTTCCGCCATTGTCGTCGGC-30 50-GGCCGTGGACACTACCCGCAACCAATCACGCAGGC-30 Experimental use RT-PCR and cloning; bridge RT-PCR; FLAG-tagged constructs RT-PCR and cloning; in vitro transcription RT-PCR and cloning; bridge RT-PCR Bridge RT-PCR Bridge RT-PCR; northern analysis FLAG-tagged constructs in vitro transcription qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR Recomb. expression Recomb. expression Recomb. expression Northern analysis Northern analysis Northern analysis RNA isolation from ﬂy tissue and cultured cells Tissues were dissected from wild-type adult ﬂies of mixed age (2- to 7-day old) in sterile 1X PBS and immediately frozen on dry ice. S2 cells were cultured under standard conditions in S2 media (Invitrogen) and collected during log phase. RNA isolation was completed using TRIzol reagent (Invitrogen) following manufacturer’s guidelines. RNase-free glycogen (10 mg; Invitrogen) was used as a carrier to increase yield. Samples with A260/280 and A260/ 230 ratios <1.8 were discarded. Qualitative analysis of 300 ng total RNA via gel electrophoresis ensured RNA integrity. Only samples that passed both spectrophotomet- ric and qualitative analyses were used for further experiments. RT-PCR Analysis Total RNA (500 ng) from adult tissues and cultured S2 cells or 100 ng of embryo, larval and adult polyA+ RNA (Clontech) was used in RT-PCR analyses. SuperScript One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen) was used following manufacturer’s guidelines. Cloning and Sequencing of rpL22-like cDNAs Gel-puriﬁed RT-PCR products were cloned into pMT/ V5-His-TOPO (Invitrogen), transformed into One Shot TOP10 Chemically Competent E. coli (Invitrogen), plated onto selective media (LB, ampicillin 100 mg/ml) and plasmid DNAs puriﬁed using the miniPrep system (Qiagen). Multiple clones for rpL22-like cDNAs were sequenced using an ABI 310 Genetic Analyzer (Applied Biosystems) or by GeneWay Research Laboratories. Bacterial recombinant protein expression for western analysis Gel-puriﬁed RT-PCR amplicons were cloned into E. coli expression vector pEXP5-CT/TOPO (Invitrogen) follow- ing manufacturer’s guidelines. Recombinant expression in transformed OneShot BL21 Star (DE3) Chemically Competent E. coli (Invitrogen) was induced with 0.5 mM IPTG. cDNA synthesis and qRT-PCR analysis Total RNA (1 mg) was used in cDNA synthesis using the SuperScript III First-Strand Synthesis System (Invitrogen) following manufacturer’s guidelines and cDNAs stored at 80 C. One microliter of cDNA was used for qRT-PCR using the ABI 7300 real-time PCR system (Applied Biosystems) with SYBR green power master mix reagent (Applied Biosystems). Primers were designed using Primer Express software (Applied Biosystems). Primers (200 nM ﬁnal concentration) were annealed at 55 C in a three-step ampliﬁcation stage. Average CT values obtained for all genes were normalized to rpL32 (rpL32/rp49) for com- parison to data by Shigenobu et al. (8). All samples were run in triplicate with an n = 3. Standard deviations for each sample were calculated using the average CT of