BMC Genomics 2007, 8:363 http://www.biomedcentral.com/1471-2164/8/363 tion was subtracted enough to remove most representation of the abundant rRNAs and greatly increase the representation of the different mRNA species. However, equalization was not complete. Since the tran- scriptome contains all intron sequences, which greatly exceed the mRNA population in complexity (at least in the zebrafish), a complete subtraction would be expected to contain far more intron than mRNA sequence. This is not the case in the libraries examined, where mRNA sequences outnumber intron sequences by a ratio of almost 7:1. We addressed the issue of equalization within the mRNA population in two different ways. We deter- mined the frequency of β-actin sequence, a particularly abundant mRNA, by probing the 200–300 bp zebrafish library with antisense oligonucleotide. Only one β-actin clone was identified among 5 × 104 colonies. Since β-actin represents approximately 1% of the mRNAs of embryonic zebrafish heart [23] and is relatively constant across tis- sues under physiological conditions [24], the representa- tion of this abundant mRNA has been reduced in the equalized libraries. In the second approach, we compared mRNA representation among our sequences with mRNA representation in an unsubtracted cDNA library con- structed from 72 hour embryonic zebrafish heart [23]. In the heart library, there are 11 mRNAs that are represented at frequencies between 0.4% (the ADP/ATP carrier pro- tein) and 4.3% (the ribosomal proteins). Of these abun- dant mRNAs, two are represented once in our collection of 240 zebrafish cDNA sequences and the rest were not represented at all. There were two other mRNA loci that were represented twice among the zebrafish self-sub- tracted sequences, one encoding Ankyrin 3 and the other encoding Ran-binding protein 2. Neither of these two loci were represented in the 5000 sequences from the heart library. There are several points of caution in using the procedure. In self-subtraction, it is the complexity of contaminating sequence, rather than its abundance, that may determine the extent of contamination in the final library. Even a few picograms of genomic DNA has a greater complexity than the entire functional RNA population. Although the importance and extent of non-coding transcripts is under revision [25] we did not observe any advantage to increas- ing the extent of self subtraction, either by including addi- tional rounds of self subtraction or by increasing the C t o value of the subtractions. Clones chosen from a fourth library that had gone through an additional round of self- subtraction and amplification did not match any mRNAs (data not shown.) The goal of the described procedure was to have as much as possible of the entire RNA population represented in a library, not to have it represented in full length copies. Some functional non-coding RNAs are almost certainly excluded from the libraries. We have used the procedure successfully on cDNA fragments as small as 100–200 bp. Self subtracted libraries of smaller sequences may be pos- sible if the phosphate concentration in the hydroxylapa- tite binding buffer is decreased and only small sequences are included in the reaction. The smallest functional proc- essed RNAs, e.g. the 22 nt microRNAs, will not be repre- sented, although their primary transcripts certainly are. Since the procedure functions via inter-molecular reasso- ciation kinetics, sequences with extensive self-homology (snap-back) will be removed independent of their abun- dance in the RNA population. The procedure will not work well for most full length transcripts since amplifying sequences larger than even 500 base-pairs in size is more difficult and more sensitive to reaction conditions. Even in our target size range of 200–300 bp there are undoubt- edly some sequences that do not amplify well by PCR and are therefore underrepresented. We believe that the more complete representation possible with short sequences offsets the disadvantage of not cloning full length copies, especially as the sequences are more than long enough to identify significant matches in the databases, encode many complete protein domains, and to serve as probes for cloning or assembling full-length cDNAs by the many other methods available. Conclusion The simple procedures described here permit the con- struction of high-quality, directional cDNA libraries using small amounts of degraded total RNA. Since the method does not distinguish between polyA+ and polyA- species, all RNAs above 100 nt may be represented, including polyA- mRNAs and many functional but non-translated RNAs. The procedure should prove valuable in situations where more complete representation of the transcriptome is desired. Methods The following procedural details are relevant to the suc- cessful use of the method. They are cited in the Results and Discussion text as (Methods-#). (1) The removal of genomic DNA contamination and purity of sample at the RNA extraction step is crucial. The self-subtraction procedure is more sensitive to the com- plexity of a contaminant than its abundance. Tissues should be processed in either disposable plasticware or baked glassware. (2) Partial hydrolysis of the RNA is an important step. The method is PCR-based and PCR efficiency becomes increasingly sequence and reaction-condition dependent as the size of the template increases. To avoid this source of bias, short cDNAs are used. Producing these by short- ening the RNA template through random hydrolysis has Page 5 of 7 (page number not for citation purposes)