The PCR-Select cDNA Subtraction Kit offers an efficient method for selectively amplifying differentially expressed genes—those genes expressed in one mRNA population but reduced or absent in another (1–4). This method is particularly well-suited for the identification of target cDNAs that correspond to rare transcripts, typically the most difficult to obtain. In contrast to other methods that require physically separating single-stranded and double-stranded cDNAs, Clontech's PCR-Select method allows the exponential amplification of only the desired sequences. This method offers many significant advantages:
- Straightforward method with only a few steps. With the PCR-Select method, subtraction occurs by one round of subtractive hybridization and selective amplification of differentially expressed genes, not by physical separation.
- Over 1,000-fold enrichment of rare transcripts. This kit allows you to equalize transcript abundance and subtract in the same procedure, dramatically increasing the probability of obtaining differentially expressed rare transcripts.
- Subtraction can be performed with just 2 µg of poly A+ RNA. This feature is especially useful when working with RNA samples that are difficult to obtain. If you have only nanograms of total RNA, generate high-quality cDNA for use in PCR-Select cDNA subtraction with the SMARTer Pico PCR cDNA Synthesis Kit.
Bacterial Genome Subtraction Kit
The Clontech PCR-Select Bacterial Genome Subtraction Kit offers an effective method for comparing bacterial genomes. In a matter of days, you can obtain a subtracted library of genomic sequences that are present in one bacterial strain but absent in another. This kit allows you to identify pathogenicity islands or other genomic DNA differences between two strains.
With the Clontech PCR-Select method, subtraction occurs in one round of subtractive hybridization and by selective amplification, not by physical separation of single-stranded DNA (1–3, 5). The Clontech PCR-Select Kit requires as little as 2 µg of each bacterial genomic DNA sample, with the procedure requiring only 2–3 days. It can be readily adapted to high-throughput sampling.
PCR-Select Differential Screening Kit
The Clontech PCR-Select Differential Screening Kit allows you to identify differentially expressed clones in a subtracted library (6–9). After generating pools of differentially expressed genes with the Clontech PCR-Select cDNA Subtraction Kit, use this kit to quickly confirm differential expression.
With the Clontech PCR-Select Differential Screening Kit, the subtracted library is hybridized with probes synthesized directly from tester and driver populations; a probe made from the subtracted cDNA, as well as a probe made from reverse-subtracted cDNA (a second subtraction performed in reverse). Clones that hybridize to tester but not driver probes are differentially expressed; however, nonsubtracted probes are not sensitive enough to detect rare messages. Subtracted probes are greatly enriched for differentially expressed cDNAs, but may give false positive results. Using both subtracted and non-subtracted probes provides the most effective way to identify differentially expressed genes.