Subtractive hybridization kits
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 (Akopyants, 1998; Jin, 1997; Diatchenko, 1996; Diatchenko, 1998). This method is particularly well-suited for the identification of target cDNAs that correspond to rare transcripts, which are typically the most difficult to obtain. In contrast to other methods that require physically separating single-stranded and double-stranded cDNAs, the PCR-Select method allows the exponential amplification of only the desired sequences. This method offers many significant advantages:
PCR-Select cDNA subtraction kits offer an efficient method for selectively amplifying differentially expressed genes—those genes expressed in one mRNA population but reduced or absent in another (Akopyants, 1998; Jin, 1997; Diatchenko, 1996; Diatchenko, 1998). This method is particularly well-suited for the identification of target cDNAs that correspond to rare transcripts, which are typically the most difficult to obtain. In contrast to other methods that require physically separating single-stranded and double-stranded cDNAs, the 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 our PCR-Select method, subtraction occurs in one round of subtractive hybridization and by selective amplification, not by physical separation of single-stranded DNA (Akopyants, 1998; Jin, 1997; Diatchenko, 1996; Diatchenko, 1998). The 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 (Diatchenko, 1998; Gurskay, 1996; Wang, 1991). After generating pools of differentially expressed genes with the Clontech PCR-Select cDNA Subtraction Kit, use this kit to quickly confirm differential expression.
With our 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 nonsubtracted probes provides the most effective way to identify differentially expressed genes.
Overview
- Ideal for isolating novel, differentially expressed genes
- Provides greater than 1,000-fold enrichment of rare transcripts
- Requires only 2 µg of poly A+ RNA
- cDNA subtraction process takes only 3–4 days
More Information
Applications
- Subtractive hybridization
- Gene identification
- Differential gene expression analysis
- Gene expression analysis
- cDNA subtraction
- Differential screening
References
Akopyants, N. S., et al. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc. Natl. Acad. Sci. USA 95:13108–13113 (1998).
Jin, H., et al. Differential screening of a subtracted cDNA library: a method to search for genes preferentially expressed in multiple tissues. BioTechniques 23:1084–1086 (1997).
Diatchenko, L., et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93:6025–6030 (1996).
Diatchenko, L., et al. In Methods in Enzymology, Ed. Weissman, S. M., 303:349–380 (1998).
Diatchenko, L., et al. In RT-PCR Methods for Gene Cloning and Analysis, Eds. Siebert, P. D., et al. (BioTechniques Books, MA), pp. 213–239 (1998).
Gurskaya, N. G., et al. Equalizing cDNA Subtraction Based on Selective Suppression of Polymerase Chain Reaction: Cloning of Jurkat Cell Transcripts Induced by Phytohemaglutinin and Phorbol 12-Myristate 13-Acetate. Anal. Biochem. 240:90–97 (1996).
Wang, Z. & Brown, D. D. A gene expression screen. Proc. Natl. Acad. Sci. USA 88:11505–11509 (1991).
Additional product information
Please see the product′s Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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