Disruption of thymopoiesis in ST6Gal I-deficient mice.

Thymocyte development is accompanied by sequential changes in cell surface glycosylation. For example, medullary thymocytes have increased levels of alpha2,3-linked sialic acid and a loss of asialo core 1 O-glycans as compared to cortical thymocytes. Some of these changes have been linked to fine tuning of the T cell receptor avidity. We analyzed ST6Gal I transcript abundance and levels of alpha2,6-linked sialic acid across thymocyte subsets. We found that ST6Gal I transcript levels increased following T cell receptor beta-selection suggesting that this sialyltransferase may influence the development of early thymocyte populations. Indeed, low levels of alpha2,6-linked sialic acid were found in the earliest T lineage cells, and then increased in T cell receptor beta-selected cells. To determine whether ST6Gal I influences T cell development, we analyzed ST6Gal I-deficient mice for disruptions in thymocyte populations. We found reduced thymic cellularity in the ST6Gal I-deficient mice starting in the early thymocyte compartments.

SAS programs for real-time RT-PCR having multiple independent samples.

Relative real-time reverse transcription PCR (RT-PCR) has become an important tool for quantifying changes in messenger RNA (mRNA) populations following differential development or stimulation of tissues or cells. However, the best methods for conducting such experiments and analyzing the resultant data remain an issue of discussion. In this report we describe an appropriate experimental methodology and the computer programs necessary to generate a meaningful statistical analysis of the combined biological and experimental variability in such experiments. Specifically, logarithmic transformations of raw fluorescence data from the log-linear portion of real-time PCR growth curves for both target and reference genes are analyzed using a SAS/STAT Mixed Procedure program specifically designed to give a point estimate of the relative expression ratio of the target gene with associated 95% confidence interval. The program code is open-source and is printed in the text.

Sialyltransferase mRNA abundances in B cells are strictly controlled, correlated with cognate lectin binding, and differentially responsive to immune signaling in vitro.

Mouse gene knockout studies have provided unimpeachable evidence of immune-relevant functions for several sialyltransferase enzymes including ST6Gal I, ST3Gal I, and ST3Gal IV. Such studies cannot, however, identify cellular mechanisms for regulating such activities. In this article we provide evidence that murine B lymphocytes respond to specific immune signals in vitro with tightly regulated changes in the sialic acid composition of the cell surface glycocalyx. These changes are both quantitative and qualitative in nature and are apparently regulated at both the transcriptional and posttranscriptional levels. We used lectin binding and flow cytometry combined with relative real-time PCR to show that MAH and PNA binding are tightly correlated with the abundance of ST3Gal IV and ST3Gal I mRNA, respectively, under several different conditions of B cell stimulation. Finally, we show that although SNA binding and the expression of ST6Gal I coding sequence are not tightly correlated, there is a clear differential control of 5'UTR exon usage in response to different immune signals.

Accurate and statistically verified quantification of relative mRNA abundances using SYBR Green I and real-time RT-PCR.

Among the many methods currently available for quantifying mRNA transcript abundance, reverse transcription-polymerase chain reaction (RT-PCR) has proved to be the most sensitive. Recently, several protocols for real-time relative RT-PCR using the reporter dye SYBR Green I have appeared in the literature. In these methods, sample and control mRNA abundance is quantified relative to an internal reference RNA whose abundance is known not to change under the differing experimental conditions. We have developed new data analysis procedures for the two most promising of these methodologies and generated data appropriate to assess both the accuracy and precision of the two protocols. We demonstrate that while both methods produce results that are precise when 18S rRNA is used as an internal reference, only one of these methods produces consistently accurate results. We have used this latter system to show that mRNA abundances can be accurately measured and strongly correlate with cell surface protein and carbohydrate expression as assessed by flow cytometry under different conditions of B cell activation.