213 SAGE ANALYSIS OF TRANSITIONS IN THE PORCINE CONCEPTUS TRANSCRIPTOME DURING TROPHECTODERM ELONGATION

Rapid elongation of the pre-implantation conceptus trophectoderm between gestational days 11 and 12 represents a critical developmental period in swine. Serial analysis of gene expression (SAGE) in ovoid (Ovd; 7 mm diameter) and filamentous (Fil; 150 mm length) conceptuses has identified unique transcriptomes associated with these transitional states. The initial utility of SAGE prompted an investigation of the intermediate tubular conceptus (Tub; 15–20 mm length) as well as the development of micro-SAGE methodologies, small-amplified RNA-SAGE (SAR-SAGE) and PCR-amplified SAGE (m-SAGE), for future use with early pre-implantation conceptus stages. Total RNA from in vivo-derived Tub conceptuses was used to construct an unamplified SAGE library (Tub SAGE) and two micro-SAGE libraries (160X less template RNA) by SAR-SAGE and m-SAGE. Comparative analyses of the amplification proportionality between the Tub SAGE and amplified libraries, consisting of 12,000 tags each, demonstrated that SAR-SAGE was a more reliable amplification method. Seventy-five percent of the SAR-SAGE tags, in contrast to 43% of m-SAGE tags, had less than a 2-fold frequency difference when compared to Tub SAGE tags occurring at least 10 times in a library. The Tub SAGE library size was extended to a total of 42,415 tags representing 12,779 unique putative transcripts. Comparing the Tub SAGE tags with previously generated Ovd and Fil SAGE libraries (NCBI GEO Acc. Nos. GSM24470 and GSM24471, respectively) yielded a total of 33,191 unique tags, of which 3,575 tags (10.8%) were common to all three libraries. Chi-square-based analyses (SAGE2000 software; 1997 Gen. Res. 7, 986–995) of the tag frequencies revealed the differential expression of 479 tags between Ovd:Tub libraries and 362 tags between Tub:Fil libraries at a P < 0.05 significance. The Tub SAGE tags were annotated following batch BLASTS against the TIGR porcine index database and then classified according to function. Real-time RT-PCR was used to confirm the differential expression patterns identified between Ovd:Tub, Ovd:Fil, and Fil:Tub. For example, the relative quantity of mRNA for steroidogenic acute regulatory protein, 1.0:6.4:11.1 (Ovd:Tub:Fil), and cytokeratin 8, 1.1:0.6:0.4 (Ovd:Tub:Fil), demonstrated an increase and decrease (ANOVA; P < 0.05), respectively, throughout these three developmental stages. Based on their putative functional annotations, the differentially expressed genes revealed by SAGE are potentially involved in regulating embryo growth, cellular differentiation, apoptosis, steroidogenesis, and energy metabolism during embryo elongation. The present SAGE analyses have elucidated the tubular conceptus transcriptome and enabled the identification of genes that are differentially regulated during the 24-h period that encompasses pig embryo elongation. Furthermore, the decreased template requirement of SAR-SAGE makes this technique a suitable option for SAGE analyses of early stage embryos where the sample is small and limited.

Statistical evaluation of SAGE libraries: consequences for experimental design

Since the introduction of serial analysis of gene expression (SAGE) as a method to quantitatively analyze the differential expression of genes, several statistical tests have been published for the pairwise comparison of SAGE libraries. Testing the difference between the number of specific tags found in two SAGE libraries is hampered by the fact that each SAGE library is only one measurement: the necessary information on biological variation or experimental precision is not available. In the currently available tests, a measure of this variance is obtained from simulation or based on the properties of the tag distribution. To help the user of SAGE to decide between these tests, five different pairwise tests have been compared by determining the critical values, that is, the lowest number of tags that, given an observed number of tags in one library, needs to be found in the other library to result in a significant P value. The five tests included in this comparison are SAGE300, the tests described by Madden et al. ( Oncogene 15: 1079–1085, 1997) and by Audic and Claverie ( Genome Res 7: 986–995, 1997), Fisher’s Exact test, and the Z test, which is equivalent to the chi-squared test. The comparison showed that, for SAGE libraries of equal as well as different size, SAGE300, Fisher’s Exact test, Z test, and the Audic and Claverie test have critical values within 1.5% of each other. This indicates that these four tests will give essentially the same results when applied to SAGE libraries. The Madden test, which can only be used for libraries of similar size, is, with 25% higher critical values, more conservative, probably because the variance measure in its test statistic is not appropriate for hypothesis testing. The consequences for the choice of SAGE library sizes are discussed.