Figure 1. (a) S1 nuclease mapping and (b) primer extension. Asterisks represent (radioactive) labels to visualize DNA fragments. The differences in band intensities, represented by thin (sample 1) and thick lines (sample 2), are directly proportional to the amount of mRNA and thus give information on the level of gene expression.

Figure 2. (a) 5¢ rapid amplification of cDNA ends (RACE) and (b) 3¢ RACE. mRNA is converted to cDNA and in the case of 5¢ RACE, an adaptor is ligated to the 5¢ end of the cDNA. Subsequently, the unknown 5¢ or 3¢ cDNA end is amplified by polymerase chain reaction (PCR) using a gene-specific primer (GSP) complementary to a known part of the gene of interest in combination with an adaptor-specific primer (ASP) (a) or a primer specific for the poly-A tail (b).

Figure 3. Suppressive subtractive hybridization. tester cDNA is divided into two equal portions and ligated to two different linkers (A, gray; B, black). Both tester portions are hybridized with excess driver cDNA resulting in tester/driver (or driver/driver) hybrids of sequences that tester and driver cDNA have in common. cDNA specific for the tester sample will not hybridize with driver cDNA. By combining both hybridized tester samples, and performing PCR using linker A- and B-specific primers, tester-specific cDNA can be amplified and cloned.

Figure 4. Differential display. All mRNAs of a cell or tissue are reverse-transcribed into cDNA using an anchored primer (5¢ (T)₁₁MN, wherein M = G, A or C and N is any base). The cDNA is divided into several pools and each pool is amplified using a pool-specific arbitrarily chosen primer and an oligo(dT) primer in the presence of labeled dNTPs. This results in the amplification of a few hundred cDNAs per pool, which are size-separated on polyacrylamide gels. Bands that are present in one sample but are not present or are weaker in the other represent upregulated genes (indicated by an arrow in sample 1) or, conversely, downregulated genes (indicated by an arrow in sample 2). By analyzing all pools of cDNAs, thousands of genes can be screened simultaneously.

Figure 5. Serial analysis of gene expression (SAGE). In SAGE, short sequence tags (approximately 10 bp) are isolated from mRNA at a defined position, ligated to long multimers, cloned and sequenced. The frequency of each tag in the cloned multimers directly reflects transcript abundancy. In addition, the short tags are long enough to uniquely identify the corresponding transcript in database searches. Thus, SAGE results in an accurate picture of gene expression at both the qualitative and the quantitative levels.