RNA extraction, cDNA synthesis Total RNA was extracted Tanespimycin from peripheral blood mononuclear cells (PBMCs) or fresh tissue using TRIzol? (Invitrogen, Carlsbad, CA, USA) or RNeasy? Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with Omniscript Reverse Transcriptase? (Qiagen) as described previously [44]. Samples were stored at -20��C. Quantification of TCR expression For determination of general TCR expression, the C��-chain was quantified on a LightCycler? instrument (Roche, Basel, Switzerland). Values were normalized using the low-abundance housekeeping gene porphobilinogen deaminidase (PBGD) as previously described [27]. For each cDNA synthesis reaction (+RT), a control reaction without reverse transcriptase was performed (-RT control). Samples with -RT/+RT ratio >0.

1 for C��-chain or PBGD indicating DNA contamination and/or RNA degradation were excluded from further analysis. The aim of our study was the detection of TCR repertoire restriction due to T-cell expansions associated with significant TCR expression of the expanded clones. Low overall TCR transcription in tissue could lead to putative oligoclonality due to the generally high sensitivity of PCR based approaches. Consequently, samples with HAC/PBGD < 0.1 were excluded from analysis to avoid this bias. Relative quantification of V��-families Relative quantification of the expression of a single TCR V��-chain was performed as described [27]. Shortly, qRT PCRs were performed with a universal reverse primer and TaqMan probe, both annealing at the constant part of the ��-chain (C��), and 28 V��-family-specific forward primers (V��-family 1 to 24, V��-families 5, 6, 12, 13 subdivided into two subgroups each).

Slope of each family specific reaction was estimated analyzing a dilution series spanning three orders of magnitude of a cDNA mixture of diagnostic samples. Calculation of the relative concentration Pj [%] of a V��-family j was carried out using the formula with Cp [amplification cycles] is the Crossing point and [��Cp/log(concentration)] is the average slope of all families. Normalization, quantification of V��-restriction The approach calculating relative concentrations of different V��-families regarding slopes and crossing points, as a matter of fact, leads to per-sample normalized values.

A per-family normalization step was added to circumvent different weighting of family alterations due to different physiological family expressions and Batimastat PCR amplification efficacies. For this purpose, the mean percentage and standard deviation SDj of every V��-family j of all analyzed samples k were determined. It was decided not only to use PBMCs from blood of healthy donors but all analyzed samples for normalization leading to more reliable results comparing repertoire restriction of tissue with blood.