Search

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.

We have developed a novel efficient approach, termed differential subtraction display, for the identification of differentially expressed genes. Several critical parameters for the reproducibility and enhanced sensitivity of display, as well as steps to reduce the number of false positive cDNA species, have been defined. These include- (a) use of standardized oligo(dT)-primed cDNA pools rather than total RNA as the starting material for differential display, (b) critical role of optimal cDNA input for each distinct class of primers, (c) phenomena of primer dominance and interference, and (d) design of a novel set of enhanced specificity anchor primers. Introduction of an efficient subtractive hybridization step prior to cloning of cDNA species enriches the bona fide cDNA species that are either exclusively present in one sample (+/−) or show altered expression (up-/down-regulation) in RNA samples from two different tissues or cell types. This approach, in comparison to differential display, has several advantages in terms of reproducibility and enhanced sensitivity of display coupled to the cloning of enriched bona fide cDNA species corresponding to differentially expressed RNAs.

The major drawback of subtractive cDNA libraries is that the original disproportion in concentrations of different types of transcripts is preserved. This usually makes the isolation of specific rare transcripts extremely difficult. To overcome this difficulty, we propose a strategy that introduces the equalization of concentrations (normalization) of specific transcripts during the subtractive process. This makes possible obtaining both rare and highly abundant transcripts in the resulting subtracted library. This technique has been applied for isolation of transcripts activated upon induction of Jurkat cells by phytohemaglutinin and phorbol 12-myristate 13-acetate. Six novel up-regulated sequences belonging to a low-abundance class of transcripts have been obtained.

Representational difference analysis of cDNAs (cDNA-RDA) is a sensitive subtractive hybridization technique capable of isolating rare mRNAs differentially expressed in two cell populations. cDNA-RDA can detect sequences represented at 0.0001% in the starting mRNA. By using reverse transcriptase polymerase chain reaction (PCR), cDNA-RDA also lends itself to studies in which samples are derived from limited numbers of cells. Standard cDNA-RDA protocols depend upon the presence of specific restriction enzyme sites in each cDNA, typically enzymes with four base recognition sequences. These sites are used to reduce the cDNA size range and provide primer sites for subsequent PCR amplification. Consequently, transcripts containing fewer than two of the chosen restriction sites are undetectable by cDNA-RDA. We have developed a restriction enzyme site-independent cDNA-RDA protocol called modified RDA (MRDA). We constructed MRDA test sequences from random hexamer-primed cDNA, thereby increasing the representation of mRNAs which are excluded by cDNA-RDA protocols. MRDA is also more efficient than cDNA-RDA at removing highly expressed housekeeping genes during the subtractive hybridization process, thereby allowing more efficient isolation of preferentially expressed mRNAs. Using MRDA, we isolated cDNAs differentially expressed between limited numbers of human CD4+ naive and memory T lymphocyte subsets and skin- and gut-homing memory T cell subsets.