Oligo dt 20 primers

RNA samples were treated with DNase I (Quiagen, Crewley, United Kingdom). Equal amounts (between 200 and 500 ng depending on the set of experiments) of total RNA were reversed transcribed using random nonamers (Sigma, Poole, United Kingdom), Oligo (dT) 20 primers (Promega), and Superscript TM III RT (Invitrogen, Carlsband, CA) for 1 hour at 50°C in a total reaction volume of 20 μL. cDNAs were immediately used for qPCR or kept at −20°C; qPCR reactions were performed with DNA Engine (BioRad, Hercules, CA) using SYBR Green JumpStart Taq ReadyMix (Sigma, Poole, United Kingdom) and gene-specific primer sets (primer sequences available on request). One microliter of cDNA diluted 1/10 in H₂O was amplified in a 3-step cycling program in a final reaction volume of 25 μL. Control cDNA samples obtained without transcriptase were always included, as well as samples without any cDNA template. Reactions were performed at least in triplicate and the specificity of the products was determined by melting curve analysis. The ratio of the relative expression of target genes to β-actin was calculated using the 2^ΔCt formula. Efficiencies of qPCRs were calculated for each gene using serial dilution.

Total RNA was isolated from the spleens of 8- to 10-week-old positive mice using an RNeasy Mini Kit (QIAGEN, Dusseldorf, Germany), and the RNA concentration was measured with Nanodrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). Reverse transcription was performed using M-MLV Reverse Transcriptase and oligo(dT) 20 primers according to the manufacturer's instructions (Promega, Madison, WI, USA). RT-PCR was used to analyse the transcription of truncated IgG1 with specific JH forward and γ1-CH2 reverse primers. Mouse GAPDH was amplified as an internal control. qRT-PCR was performed using LightCycler 480 SYBR Green I Master mix (Roche, Basel, Switzerland) with primers for IgG1, IgM and GAPDH under the following cycling conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The relative transcription levels of IgG1 and IgM were determined using the 2^−ΔΔCt method by comparing the values with the internal control GAPDH.
Furthermore, the recombined variable region sequences of the truncated IgG1 or IgM were amplified by 5′ RACE according to the manufacturer's protocol (Invitrogen, BioSource International, USA). All variable region sequences were blasted against the IMGT database (http://www.imgt.org/ligmdb/) to analyse the V, D, J, and CDR3 sequences.