characterized, silencing is known to spread in a 39 to 59 direction of the targeted mRNA [3]; hence the 59 region of the target mRNA is longer lived and more available for qRT-PCR based quantification than regions in the 39 region. For CYP6B46, CYP6B45, CYP4M1, CYP4M2 and CYPM3, the size of this amplicon was 118BP (+144 to +261), 105 bp (+613 to +718), 101 bp (+868 to +968), 166 bp (+453 to +619) and 130 bp (+703 to +832), respectively. The specificity of these primers for insect cDNA was confirmed by a PCR with N. attenuata (WT) leaf cDNA. For each sample, 500 ng of total RNA was used for cDNA preparation using oligo(dT)18 primer and SuperScript II enzyme (Invitrogen) with the manufacturer’s recommendations. All the qRT-PCRs were performed with a Mx3005P Multiplex qPCR system (Stratagene) and the qPCR core kit for SYBR Green I (Eurogentec). Relative quantification of mRNA levels was performed by the comparative D cycle threshold (CT) method using the MsUbiquitin mRNA as an internal standard. All the qPCRs were performed using the following conditions: initial denaturation step of 95uC for 30 s, followed by 40 cycles each of 95uC for 30 s and 60uC for 1 min, with a final extension step of 95uC for 30 s and 60uC for 1 min. All the results were obtained from at least five independent biological replicates and two technical replicates. Plant transformation Stable transgenic inverted repeat-MsCYP6B46 (ir-CYP6B46) N. attenuata lines were generated by transforming the recombinant pSOL8 transformation vector [28]. Vector contained 300 bp fragment of M. sexta CYP6B46 gene in an inverted repeat orientation along with the hygromycin phosphotransferase (hptII) gene providing hygromycin resistance as a selectable marker (Fig. S2A) [41]. Screening of the transgenic lines followed the protocol recommended by Gase et al. [42]. Two homozygous independently transformed ir-CYP6B46 lines (30-2 and 416-3) harboring single insertions of the hptII marker gene were used for further studies. Wild type (WT) and the stable transgenic empty pSOL8 vector (EV) containing [43] plants were used as negative controls. Southern Hybridization A modified cetyltrimethylammonium bromide method de- scribed by Rogers and Bendich [44] was followed for genomic DNA extraction from the fully expanded rosette leaves of WT, ir- CYP6B46 (30-2) and ir-CYP6B46 (416-3) N. attenuata plants. For Southern-blot hybridizations 10 mg of the genomic DNA was completely digested with HindIII and EcoRV, separately. It was then size fractionated on 1% (w/v) agarose gel, and blotted onto a nylon membrane (GeneScreenPlus; Perkin-Elmer) by capillary transfer. Hybridization and detection of the insertion number was performed as described by Gase et al. [42]. Virus induced plant gene silencing (VIGS) A VIGS system based on the TRV was used to silence N. attenuata DCLs, as described by Saedler and Baldwin [29]. VIGS vector (pTV) harboring $300 bp fragments of NaDCL1 (JN032013), Na-DCL2 (JN032015), NaDCL3 (JN032015), and NaDCL4 (JN032016) (Bozorov et al., in review) were used for VIGS. A vector without insert (EV) was used as a negative control. To silence the four NaDCLs in various combinations, the Agrobacterium cultures containing respective DCL-VIGS constructs were mixed in equal proportions before infiltration in three ir- CYP6B46 (30-2) plants. Primers were designed to amplify 102 bp of the cloned and expressed MsCYP6B46 dsRNA in leaf (Fig. S4A; Table S1). Plants with DCL-VIGS combination retaining higher levels of 102 bp MsCYP6B46 transcripts were detected by qRT- Plant Mediated RNAi Silencing Manduca Sexta Genes PCR and were selected for feeding M. sexta larvae. Silencing of each NaDCL used in the selected VIGS combinations was confirmed by the qRT-PCR based transcript quantification (Fig S4B- S4E). Prior to this work, it was confirmed that the primers used for NaDCL transcript quantification (Table S1) produced a single amplicon with N. attenuata (WT) cDNA (Fig S4F). Plant tissue, RNA isolation, cDNA synthesis and qRT-PCR Fully expanded N. attenuata rosette leaves were used in the entire analysis. They were harvested and immediately frozen in liquid nitrogen. The protocols used for insect tissues were also followed for their RNA isolation, cDNA synthesis and qRT-PCR. NaActin was used as an internal standard for qRT-PCR. All the results were obtained from three independent biological replicates and two technical replicates. Plant-Virus based dsRNA Producing System (VDPS) N. attenuata VIGS system was modified with the substitution of inserted plant cDNA fragment by the insect cDNA fragment. Selected $300 bp fragment of MsCYP6B46 or MsCYP4M1 or MsCYP4M3 was cloned in the pTV vector in an antisense orientation (Fig. S3) and Agro-infiltrated into WT plants to express $300 bp dsRNA of the respective cloned fragment. An empty pTV vector (EV) was used as a negative control. These transformed plants were fed to M. sexta larvae to silence the expression of MsCYP6B46, MsCYP4M1 and MsCYP4M3, respectively. Low Molecular Weight (LMW) RNA isolation For the isolation of low molecular weight (LMW) RNA, the total RNA (from insect midgut as well as from leaves) was subjected to PEG (10%) precipitation in presence of 1 M NaCl. Subsequently, it was precipitated using isopropyl alcohol (0.8 volumes) by incubating overnight at 220uC. Northern blotting and hybridization for the detection of smRNA Fifty mg LMW RNA (pool of three biological replicates from insect midguts or leaves) was separated under denaturing conditions (8 M urea) in 15% acrylamide gel, stained with ethidium bromide and visualized with the help of a UV transilluminator. Similar fluorescence intensity of the 5.8 S rRNA bands reflected the equal loading of LMW RNA. The RNA was transferred to a nylon membrane (GeneScreenPlus; Perkin-Elmer) by electro-blotting at 400 mA for 1 h, using 0.56 TBE as a transfer buffer. Fragments of MsCYP6B46 were PCR amplified using primer pair #4 (Table S1) for the preparation of the probe. Ten ng of these amplicons were labeled with a-32P using the Rediprime II DNA labeling system (Amersham Biosciences). Hybridization and screening of the blot performed as previously explained, except that the blots were exposed to the Fujifilm (www. fujifilm.com) BAS-MS imaging plates for 10 to 12 days until no further increase in signal was observed [45]. Statistical analysis All statistical analyses were performed with StatView version 5 (SAS Institute Inc.). Significance of variance was determined after the one way ANOVA (P.0.05) and was represented in all the graphs as 6S.E. Supporting Information Figure S1 Alignments of MsCYPs cDNA regions selected for cloning; insect cDNA specific amplification by qRT- PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31347