T cell receptor BV repertoires using real time PCR: a comparison of SYBR green and a dual-labelled HuTrec fluorescent probe.
Real time PCR is a useful tool in immunological research but little has been published on the use of this technique in the measurement of T cell receptor (TCR) BV repertoires. We have compared the performance of SYBR green with that of a dual-labeled HuTrec (Human T cell receptor) fluorescent probe system. Serial dilutions of peripherals blood mononuclear cells were tested to compare the consistency of the two systems across multiple T cell receptor signal levels. Samples were diluted with non-TCR cDNA to simulate a low-level TCR signal within a tissue sample. The fluorogenic probe gave highly consistent results with a correlation coefficient of greater than 0.9 across a samples. SYBR green showed accurate results only when tested with high signal samples. Low-signal cDNA gave very poor results with SYBR green compared to the HuTrec fluorogenic probe with correlation coefficients as low as 0.65. Poorest performance occurred in the context of the simulated tissue sample with a high level of non-TCR DNA. Under these conditions, large amounts of nonspecific PCR product were generated which were detected by the SYBR green system and therefore distorted the results. SYBR green performed poorly when used with samples contaminated with significant quantities of non-target cDNA and samples with low target signal. It is therefore not an appropriate method for the measurement of TCR repertoires in small tissue samples. A dual-labeled HuTrec fluorescent probe produced a consistent TCR repertoire across a broad range of TCR signal levels and proved robust in the presence of contaminating non-TCR cDNA. We recommend the use of such a fluorescent probe in real time PCR for the assessment of TCR repertoires in small tissue samples. Where samples are assayed using SYBR green, agarose gel confirmation of PCR product specificity should be provided.
Walters
Giles
G
Department of Nephrology, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, United Kingdom. gilesw@doctors.net.uk
Alexander
Stephen I
SI
D052160:Benzothiazoles; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D003959:Diamines; D015329:Gene Rearrangement, T-Lymphocyte / Q000235:genetics; D019674:Genes, T-Cell Receptor beta / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry*; D011804:Quinolines; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D013223:Statistics as Topic
D052160:Benzothiazoles; D017931:DNA Primers; D018076:DNA, Complementary; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases