Subtractive hybridization is a powerful technique particularly well-suited for the identification of target cdnas that correspond to rare transcripts, that are expressed in one population but not in the other. This method allows the exponential amplification of only the desired sequences, comparing two populations of mrna. cdna is synthesized from 0.5-2 g of poly A+RNA from the two types of tissues or cells being compared. The tester and driver cdnas are digested with RsaI (4bp). The tester cdna is then subdivided into two sub-samples, and each is ligated with a different cdna adaptor. The two adaptors have stretches of identical sequence to allow anneling of the PCR primer. Two hybridizations are then performed. Then an excess of driver is added to each sample of tester. The samples are then heat-denatured and allowed to anneal, generating the type a, b, c, and d molecules in each sample. Only the remaining equalized and subtracted ss tester cdnas can reassociate and form new type e hybrids. These new hybrids are ds tester molecules with different ends, which correspond to the sequences of Adaptor1 an 2R. Fresh denatured driver cdna is added to further enrich fraction e for differentially expressed sequences. Fill in the ends by DNA polymerase, the type e molecules -the differentially expressed tester sequences- have different annealing sites for the nested primers on their 5' and 3'ends.
The cdna in which specific transcripts are to be found is referred to as tester and the reference cdna is referred to as driver. First, both mrna populations are converted into cdna: we refer to the cdna that contains specific (differentially expressed) transcripts as tester, and the reference cdna as driver. Tester and driver cdnas are hybridized, and the hybrid sequences are then removed. Consequently, the remaining unhybridized cdnas represent genes that are expressed in the tester yet absent from the driver mrna.
GTAC CATG RsaI Denature & Annealing Denature & Annealing Type e molecules are formed only if the sequence is upregulated in the tester cdna. Solid lines represent the Rsa I-digested tester or driver cdna. Solid boxes represent the outer part of the Adaptor 1 and 2R longer strands and corresponding PCR primer 1 sequence. Clear boxes represent the inner part of Adaptor 1 and the corresponding Nested PCR primer 1 sequence. Shaded boxes represent the inner part of Adaptor 2R and the corresponding Nested PCR primer 2R sequence.
Tissues were snap frozen in liquid nitrogen immediately after surgical resection and stored at 80 C. For isolation of total RNA, serial cryosections were directly dissolved in 4 M guanidinium thiocyanate (denatura proteine, inattiva RNasi) containing 1% -mercaptoethanol, and the lysate was subjected to ultracentrifugation over a CsCl gradient Poly(A) RNA (mrna) was extracted using the Oligotex Direct mrna kit To test cdna samples for residual genomic DNA, PCR was performed with 0.1 μg of cdna and primers specific for an intronic region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) All libraries were generated by suppression subtraction hybridization (SSH), using poly(a) RNA of tumor tissue or a tumor cell line as a tester, and the corresponding normal tissue or a pool of normal tissues or a primary cell line as a driver.
For the preparation of our subtractive libraries, we have developed and applied a protocol for the generation of cdna fragments with increased size. When following the original protocol relying on a restriction enzyme recognizing only a 4-base motif such as RsaI, we have observed a high percentage of fragments to be smaller than 50 bp. Accordingly, we have used a set of 6-base recognizing restriction enzymes, 3 with A/T-rich (primarily found at the 3 -end of eukaryotic cdnas) and 3 with G/C-rich recognition sequences (characteristic for 5 -termini of eukaryotic genes). Our approach resulted in a considerable shift toward longer cdna fragments; sequence analysis of several thousands of clones revealed an increase in average length to 800 bp. Such longer cdna fragments are more favorable for cdna microarrays
PCR products of subtractive lung squamous cell carcinoma cdna libraries generated either by a 4- base or a pool of 6-base recognizing restriction enzymes. Lanes 1 and 4: DNA size marker ( x/haeiii + /HindIII); Lane 2: subtractive library generated using a pool of 6-cutters; Lane 3: subtractive library generated using the 4-cutter RsaI; arrows indicate the respective position for keratin 6A cdna fragment.
Drawing representing the developmental steps of flower development in saffron. At the beginning of September, one or more shoots emerge from the underground corm with the undeveloped flowers and leaves wrapped by the cataphylls, as flower develops the cataphyll grows protecting the flower up to the moment of shot emergence from the soil, which takes place during October- November, when the flower is complete developed. (B) Schematic representation of the apocarotenoid biosynthetic pathway in saffron stigma.
Suppression subtractive hybridization (SSH) is a powerful technique for the identification of differentially expressed genes, including those genes present in relatively low abundance. We used SSH to isolate and characterize expressed sequence tags (ESTs) produced in red stigmas in response to light exposure during 24 hours (tester) versus stigmas submitted to 24 hours of darkness (driver). Among the identified cdna fragments, two of them showed the highest levels of induction by light and were chosen for further analyses and characterization.