After purification, the RNA concentration was measured with a Nanodrop® spectrophotometer (Thermo Scientific, Wilmington, DE) and the RNA quality was checked on an agarose gel electrophoresis. Reverse-transcription into the first cDNA strand was carried out using the First strand Synthesis System for the RT-PCR kit (Invitrogen, Cergy-pontoise, France). Real-time RT PCR transcript quantification Quantitative measurements were performed on RNA samples originating from 5 independent replicates.
Quantification was performed with a LightCycler®480 system using the Selleckchem Adavosertib LightCycler Fast Start DNA Master SYBR green I kit (Roche Diagnostics, Meylan, France). Data were normalized using GDC-0068 mouse the ratio of the target cDNA concentration to that of the glyceraldehyde 3-phosphate dehydrogenase (gapdh) gene and the ribosomal check details protein L29 (RPL29) gene. Primers were designed to amplify fragments with less than 250 bp and are listed in the additional file 1. The PCR reactions were carried out in LightCycler 96-well plates, in a final volume of 10 μl, containing 2.5 μl of cDNA samples (diluted five-fold) and 7.5 μl of Light Cycler® 480 SYBR Green Master 1 mix, together with 0.5 μl of 10 mM of each primer, 1.5 μl H2O and 5 μl of Mastermix. Quantification was realized as described by . Normalization and statistical pair-wise comparisons were determined using REST . When comparing more than two
modalities at the same time, the non-parametric Kruskal-Wallis test was used. RPL29 was shown to be the best housekeeping gene, with Bestkeeper tool , and this has been used in graphical representations. Results General characteristics of libraries: 8,941 weevil unigenes were generated To explore bacteriome cellular specificities and weevil immune responses to bacteria, we have constructed 7 cDNA libraries from S. oryzae larvae. These libraries comprise the 4 SSH libraries, SSHA, SSHB, SSH1
and SSH2, the 2 non-normalized libraries from symbiont-full (SO) and symbiont–free (AO) bacteriomes and one normalized library (NOR) from http://www.selleck.co.jp/products/Staurosporine.html whole aposymbiotic larvae challenged, and not, with S. typhimurium (Fig. 2A). Figure 2 General description of libraries. (A) Table of ESTs and Unigene numbers presented for each library. The percentages of mitochondrial and rRNA sequences are also provided. (B) Distribution of unigenes (UGs) as a function of the number of ESTs involved in the UG sequences. UGs with only one EST are singletons, UGs with more than one EST are contigs. (C) Blast2go annotation results. Number of sequences presenting GO terms association is given for each step of the functional annotation. The different steps are described in the Methods section. The sequencing of all the libraries has generated 26,886 readable ESTs with sequence mean lengths of 520 ± 177 bp. Contigation analysis has generated 8,941 unigenes.