Original Pool Research Articles

Exploratory Polysomnographic Evaluation of Pregabalin on Sleep Disturbance in Patients with Epilepsy

To evaluate the effects of adjunctive pregabalin 300 mg/day versus placebo on polysomnographic (PSG) variables in patients with well controlled partial seizures and subjectively reported sleep disturbance. An exploratory, 4-week, double-blind, randomized study in patients with well controlled partial seizures on AED monotherapy and subjective sleep disturbance over the previous 6 months. Mean changes from baseline to endpoint in PSG and subjective sleep variables (MOS Sleep Scale, Groningen Sleep Questionnaire) in patients on adjunctive pregabalin 300 mg/day (n=8) were compared with patients on placebo (n=7). Baseline PSGs showed sleep fragmentation. Mean sleep efficiency improved significantly in both treatment groups in the mean baseline to endpoint change; there was no significant between-group difference. Pregabalin treatment was associated with a significant reduction in number of awakenings (p = 0.02), and improvement in wake time after sleep onset approached significance (p = 0.055), suggesting improvement in sleep continuity that was not observed in the placebo group. Pregabalin was also associated with significant improvements in the MOS sleep disturbance and sleep quantity subscales compared with placebo (p < or =0.03). There were no changes in self-reported seizure control. This exploratory pilot study suggests that pregabalin may improve sleep continuity in patients with clinically relevant sleep disturbance. The effect on disturbed sleep appears independent of seizure control. The effects of pregabalin on disturbed sleep and seizures and their interrelationships warrant further study.

Quantitative Characterization of Regenerating Axons after End-to-Side and End-to-End Coaptation in a Rat Brachial Plexus Model: A Retrograde Tracer Study

The efficacy of end-to-side repair as a method of nerve reconstruction has been questioned, and most studies that characterize the mode of re-innervation are marred by inappropriate experimental design and lack quantitative analysis. This makes characterization of re-innervating neurons confusing and consequently controversy remains as to the extent and source of reinnervating axons. In an experimental brachial plexus rat model, we transected the musculocutaneous nerve, labeled its neuron pool with Fast-Blue and joined the distal stump to the side of the intact ulnar nerve, or to the proximal stump of the divided ulnar nerve, to characterize neurons that reinnervate the recipient nerve. Tetramethyl-rhodamine dextran (TMRD) or fluoro-gold was used to map the reinnervating motor and sensory neurons at 12 weeks post-transection. No neurons originally labeled from musculocutaneous nerve were subsequently labeled with TMRD or fluoro-gold, showing that this original neuron pool does not contribute to re-innervation of the distal musculocutaneous nerve, but that reinnervation occurs solely by ulnar nerve motor and sensory axons. In the end-to-side group, 16.4% of the motor and 7% of the sensory donor ulnar nerve neurons re-innervated the musculocutaneous nerve exclusively, and a further 10% motor and 11.6% sensory innervated the musculocutaneous nerve by collateral sprouting of their axons. This compared to re-innervation by 62.6% of motor and 70.4% of ulnar nerve sensory neurons in the positive control that underwent end-to-end repair. Our results confirm the concept of collateral sprouting and support the use of end-to-side repair.

Development and Testing of an Evaluation Scale of Personal Digital Assistants

With the increasingly popular use of PDAs in healthcare, the need for a reliable scale to evaluate this technology has become critical. The authors describe the development and testing of an evaluation scale for PDAs in nurses' daily practice. A review of the literature generated a 30-item questionnaire that was administered online to a convenience sample of 334 hospital nurses in Taiwan. The scale's reliability and validity were established by item and factor analyses. From the original pool of 30 items, 24 were selected and grouped into six major constructs: user preference, nursing efficiency, user training, device features, drug content, and user support. The scale's overall reliability (alpha coefficient) was 0.88. Survey results showed an overall slightly negative score for the evaluation scale for PDAs, indicating a slightly moderate evaluation of PDA use. Additional psychometric analysis and refinement of item wordings are recommended.

An empirical study on selective partitioning dimensions for partition-based similarity joins

Real-world application data are usually distributed sparsely and non-uniformly in the high dimensional space that is huge in size. Hence, selection of effective partitioning dimensions is crucial for partition-based similarity joins. In this paper, we present two data partitioning algorithms for evaluations. PerDimSelect selects some dimension axes from the original perpendicular dimension axes pool, and maps each data point into the reduced dimension space. DiaDimSelect creates one-dimensional axis by combining some of original perpendicular dimensions, and maps each data point into the newly-created dimension. In the experiments, several measures are used to compare the performances of the algorithms including CPU cost, total response time, number of created buckets. In conclusion, DiaDimSelect shows better performance than PerDimSelect, for it creates much less partition buckets with the increasing number of partitioning dimensions, which leads to keep the IO cost less expensive while decreasing CPU cost considerably.

A measure of satisfaction with food-related life

A measure of satisfaction with food-related life is developed and tested in three studies in eight European countries. Five items are retained from an original pool of seven; these items exhibit good reliability as measured by Cronbach's alpha, good temporal stability, convergent validity with two related measures, and construct validity as indicated by relationships with other indicators of quality of life, including the Satisfaction With Life and the SF-8 scales. It is concluded that this scale will be useful in studies trying to identify factors contributing to satisfaction with food-related life.

Development and validation of the Generalized Anxiety Disorder Inventory (GADI)

The psychometric tools used for the assessment of generalized anxiety disorder (GAD) either do not conform to the current concept of the condition or have important limitations. We aimed to develop and validate a new questionnaire for the assessment of symptom profile and severity of GAD. An original pool of potential scale items was subjected to a series of studies in non-clinical and clinical populations, in order to determine the final composition of the scale. The psychometric properties of the new scale, the Generalized Anxiety Disorder Inventory (GADI), were evaluated using a factor analytic model suitable for ordinal data and the Graded Response Model. The precision of measurement of the GADI was quantified through the item information functions.A total of 197 outpatients and 522 non-clinical subjects participated in four studies and completed the GADI. The final 18-item scale was derived from an original pool of 30 potential items. The GADI showed good reliability, convergent and divergent validity. The scale comprises three factors, relating to cognitive, somatic and sleep symptoms. It accurately distinguished GAD patients from non-patient controls. The cognitive factor also distinguished GAD from other anxiety disorders and depression. The GADI is a useful tool in the assessment of the breadth of symptoms and the severity of generalized anxiety disorder in clinical settings.

A mathematical analysis of SELEX

Systematic evolution of ligands by exponential enrichment (SELEX) is a procedure by which a mixture of nucleic acids that vary in sequence can be separated into pure components with the goal of isolating those with specific biochemical activities. The basic idea is to combine the mixture with a specific target molecule and then separate the target–NA complex from the resulting reaction. The target–NA complex is then separated by mechanical means (for example by filtration), the NA is then eluted from the complex, amplified by polymerase chain reaction (PCR) and the process repeated. After several rounds, one should be left with a pool of [NA] that consists mostly of the species in the original pool that best binds to the target. In Irvine et al. [Irvine, D., Tuerk, C., Gold, L., 1991. SELEXION, systematic evolution of nucleic acids by exponential enrichment with integrated optimization by non-linear analysis. J. Mol. Biol. 222, 739–761] a mathematical analysis of this process was given. In this paper we revisit Irvine et al. [Ibid]. By rewriting the equations for the SELEX process, we considerably reduce the labor of computing the round to round distribution of nucleic acid fractions. We also establish necessary and sufficient conditions for the SELEX process to converge to a pool consisting solely of the best binding nucleic acid to a fixed target in a manner that maximizes the percentage of bound target. The assumption is that there is a single nucleic acid binding site on the target that permits occupation by not more than one nucleic acid. We analyze the case for which there is no background loss (no support losses and no free [NA] left on the support). We then examine the case in which such there are such losses. The significance of the analysis is that it suggests an experimental approach for the SELEX process as defined in Irvine et al. [Ibid] to converge to a pool consisting of a single best binding nucleic acid without recourse to any a priori information about the nature of the binding constants or the distribution of the individual nucleic acid fragments.

Common bean (Phaseolus vulgaris L.) landraces in Catalonia, a Mesoamerican germplasm hotspot to be preserved

SummarySeveral landraces of common bean with high organoleptic value have resisted the rapid expansion of improved cultivars in Catalonia, in north-eastern Spain. To establish strategies for their preservation and use, we employed RAPD and AFLP markers to investigate the genetic variability within 15 landraces and to identify their original gene pools. A higher percentage of Mesoamerican landraces was found in Catalonia (40%) than in the rest of the Iberian Peninsula, or in Europe (approx. 20%). This is probably due to the exclusion of Catalonia from early trade with the American colonies and stronger commercial links with the Caribbean during the nineteenth century. Our results confirm that Catalan consumers prefer white-seeded common bean varieties of Mesoamerican origin. The landrace ‘Castellfollit del Boix’ showed 69.6% polymorphic RAPD primers, with 53.2% polymorphic bands, while at the other extreme ‘Tavella Brisa’ showed 47.8% polymorphic primers with 25.3% polymorphic bands. An AFLP approach yielded similar results. The high genetic variability found in ‘Castellfollit del Boix’, one of the landraces most threatened, suggests a considerable amount of introgression from improved inbreds.

The State of Qualitative Research in Gifted Education as Published in American Journals

As qualitative research has become a more familiar form of inquiry in gifted education, judging its quality and value remains obscure and problematic to the field. This article analyzes and critiques published studies for the purpose of understanding the state of qualitative research in gifted education. Data for this study are from the major American gifted education journals. Publications between 1985 and 2003 are surveyed, electronically and manually, to form the pool of 124 qualitative studies. Each study is summarized, analyzed, categorized, and evaluated based on the attributes of qualitative research. Of the original pool, 40 are consistent with the qualitative paradigm; others are judged as qualitative lite or examples of quantitative research. Exemplars of quality studies are presented as referents for readers to further their understanding and investigation of the quality and value of qualitative research.

Turbidimetric carbamazepine immunoassay on the ADVIA® 1650 and 2400 analyzers is free from interference of antihistamine drugs hydroxyzine and cetirizine

A recent report indicates that hydroxyzine and its active metabolite cetirizine interfere with the particle-enhanced turbidimetric inhibition immunoassay (PENTINA) carbamazepine assay. We studied potential interference of hydroxyzine and cetirizine with the turbidimetric carbamazepine immunoassay on ADVIA 1650 and ADVIA 2400 (Bayer Diagnostics, Tarrytown, NY) analyzers. Aliquots of drug-free serum pools were supplemented with various concentrations of hydroxyzine and cetirizine representing therapeutic, moderate toxic, as well as very toxic concentrations. These samples were assayed by the turbidimetric carbamazepine immunoassay on two analyzers. To study the interference in presence of the analyte, aliquots of a serum pool prepared from patients receiving carbamazepine were further supplemented with various amounts of hydroxyzine and or cetirizine and apparent carbamazepine concentrations were measured again in order to compare with the value of original pool. No apparent carbamazepine concentration was observed when aliquots of drug-free serum were supplemented with hydroxyzine or cetirizine. Moreover, in the carbamazepine pool, the original carbamazepine concentration compared well when aliquots of this serum pool were further supplemented with hydroxyzine or cetirizine. We conclude that the turbidimetric carbamazepine immunoassay is free from interference of hydroxyzine and cetirizine.

Effects of a Single Foot Rot Incident on Weight Performance of Feedlot Steers

AbstractFeedlot performance records from the U.S. Meat Animal Research Center feedlot for 1993 through 2000 were analyzed to evaluate the impact of foot rot on ADG and total days on feed. Records from the original pool of 36,755 bull, steer, and heifer calves were sorted so that only steers that had a single foot rot incidence and no other morbidities were included in the data set (7,100 steers). To roughly pattern these data to industry production practices, time of foot rot insult during feeding was divided into 3 production periods: starting (0 to 60 d), growing (61 to 120 d), and finishing (121 d to harvest). Records were evaluated to determine which limb was more likely to be affected with foot rot. A total of 459 (6.5%) steers were treated for a single foot rot incident. The ADG for cattle not affected by foot rot was 1.30kg. For cattle experiencing a single foot rot incident, the ADG was 1.27kg (P = 0.03). The production period of foot rot onset impacted both ADG and total days on feed. Steers diagnosed with foot rot during the starting period gained 0.032 kg/d more (P = 0.083) than non-affected steers. Steers diagnosed in the growing and finishing periods gained 0.009 and 0.049 kg/d less than non-affected cattle (P = 0.438 and P < 0.01, respectively). Mean days on feed for the non-affected cattle was 262 d and mean days on feed for foot rot-affected cattle was 267 d (P < 0.01). The impact of foot rot on days on feed for periods 1 through 3 was −9.9 d, +2.2 d, and +14.3 d (P < 0.01, P = 0.26, P < 0.01, respectively). Foot rot diagnosed in either front limb reduced (P = 0.014) BW gain by 0.031kg.

New Enzyme-Linked Chemiluminescent Immunosorbent Digoxin Assay Is Free From Interference of Chinese Medicine DanShen

DanShen is a traditional Chinese medicine indicated for cardiovascular diseases. The potential interference of DanShen with serum digoxin measurement was investigated using a new enzyme-linked chemiluminescent immunosorbent (ECLIA) digoxin assay. Aliquots of drug-free serum were supplemented with ethyl acetate extract of DanShen (4 different brands studied), and apparent digoxin concentrations were measured by the ECLIA as well as fluorescence polarization immunoassay (FPIA) and a turbidimetric assay for comparison. Mice were also fed 4 DanShen preparations and apparent digoxin concentrations were subsequently measured. In another experiment, serum pools containing digoxin were further supplemented with DanShen extracts and digoxin concentrations were measured again by all 3 assays. No apparent digoxin concentration was observed when aliquots of drug-free serum pools were supplemented with DanShen and digoxin concentrations were measured by the ECLIA or the turbidimetric assay. In contrast, significant apparent digoxin concentrations were observed using FPIA, and the highest apparent digoxin concentration was observed with brand 4 of DanShen extract. Similarly, when mice were fed with this herb, significant apparent digoxin concentrations were also observed using FPIA, but neither ECLIA nor turbidimetric assay showed any apparent digoxin concentration. When aliquots of digoxin pool were further supplemented with various DanShen extract, the apparent digoxin concentrations were significantly increased when FPIA was used. In contrast, digoxin concentrations in the presence of DanShen extract compared well with the digoxin concentration of the original pool when ECLIA or turbidimetric assay was used. We conclude that DanShen does not interfere with serum digoxin measurement using a more recently released ECLIA digoxin assay.

Psychometric Properties of the Reidenbach–Robin Multidimensional Ethics Scale

The factor structure of the Multidimensional Ethics Scale (MES; Reidenbach and Robin: 1988, Journal of Business Ethics 7, 871–879; 1990, Journal of Business Ethics 9, 639–653) was examined for the 8-item short form (N = 328) and the original 30-item pool (N = 260). The objectives of the study were: to verify the dimensionality of the MES; to increase the amount of true cross-scenario variance through the use of 18 scenarios varying in moral intensity (Jones: 1991, Academy of Management Review 16, 366–395); and, to examine the items for measurement precision using item-response theory (IRT) methods. Results of confirmatory and exploratory factor analysis failed to conclusively support the hypothesized 3- (short form) or 5-factor (long form) structure; both instruments were instead dominated by a general factor. Item response theory analyses using Samejima’s (1969, Psychometrika Monograph Supplement 34, (4, Pt. 2)) graded response model revealed that many items in the 30-item pool performed very well, and suggested that a different collection of items be used to form a short-form version of the MES. Our proposed 10-item instrument includes more discriminating items than the 8-item version, and has the added advantage of including two items from each of the five ethical philosophies represented in the original 30-item pool.

Development and Refinement of a Measure of Attitudes Toward Sex Offender Treatment

In recent years public attitudes toward sex offenders have become increasingly punitive. Consequently, new legislation pertaining to the sentencing and treatment of convicted sex offenders has been focused on containment and monitoring rather than rehabilitation. However, research suggests that treatment programs for sex offenders are effective in decreasing subsequent sexual recidivism. This study describes the development and refinement of a brief scale for assessing public attitudes toward the treatment of sex offenders (ATTSO). Of the original item pool, 15 items were found to statistically and theoretically function well, forming three internally consistent factors measuring attitudes of incapacitation, treatment ineffectiveness and mandated treatment. The utility of the scale as it pertains to treatment centers and public policy development will be discussed.

Diversity and natural hybridization in a highly endemic species of Petunia (Solanaceae): a molecular and ecological analysis

Intrinsic reproductive barriers among the species of Petunia are weak and genetic isolation is obtained mainly by geographical separation and ecological diversification. The Serra do Sudeste region in the extreme south of Brazil is one of the centres of diversity of this genus and is characterized by the presence of species with different pollination syndromes. Petunia exserta is known only from four sandstone towers in a restricted area of this region (about 500 km(2)) and is characterized by its differentiated habitat (shelters in the sandstone towers) and by its floral characteristics adapted to ornithophily. In towers where this species is sympatric with the sphingophilous Petunia axillaris, phylogenetically close to P. exserta, we found plants with intermediate floral morphology, suggesting hybridization between them. To test this hypothesis and to better understand its consequences we analysed the sequences of the plastid trnH-psbA, trnS-trnG and psbB-psbH intergenic spacers in 121 individuals sampled all over the P. exserta distribution. The joint analysis of the three markers revealed 13 haplotypes and the network showed two main genetic clades, which probably represent the original gene pool of the two species in the region. In general, individuals of a given population presented the same haplotype, independently of phenotype, corroborating the hybridization hypothesis. Field observations suggest that hummingbirds are responsible for the interspecific gene flow. Analysis of molecular variance revealed high interpopulational diversity among the towers. The low gene flow between populations is possibly related to the autochoric seed dispersion system.

Age at Natural Menopause in Women Exposed to Diethylstilbestrol in Utero

TM1-O-03 Introduction: A woman's age at natural menopause is related to several important health outcomes, including breast cancer, cardiovascular disease, and overall mortality. Age at menopause may be affected by the size of the original pool of follicles formed during gestation, suggesting that in utero factors could be important in determining menopausal age. Diethylstilbestrol (DES), a nonsteroidal estrogen widely prescribed during the 1950s and 1960s to prevent miscarriage, has been related to numerous reproductive tract abnormalities and adverse health outcomes among prenatally exposed daughters but has not been studied previously in relation to age at menopause. Methods: We analyzed age at natural menopause in 4 combined cohorts of 4210 women exposed to DES in utero, and a comparable group of 1829 nonexposed women, all of whom had exposure status documented by prenatal medical records. The original cohorts were first identified in the 1970s from several different geographic regions in the United States. Women were recontacted by mail questionnaire in 1994, 1997, and 2001. Questions were asked about the occurrence of natural or surgical menopause, hormone replacement therapy, and other risk factors. Survival analysis methods, including Cox proportional hazards modeling, were used to estimate the risk of natural menopause in DES-exposed versus unexposed women. Results: DES-exposed women were approximately 50% more likely to be naturally menopausal than unexposed women at any given age (hazard ratio [HR] = 1.48; 95% CI, 1.28–1.71), controlling for year of birth and original cohort. Adjustment for a broad range of other confounders had little effect on the results. On average, DES-exposed women reached menopause 11 months earlier than unexposed women, which, in our data, was approximately the same effect size as having ever smoked. In a subset of women with information on the total cumulative dose of DES, dose-response effects were found, with a twofold increased risk of menopause among women exposed to >10,000 mg during pregnancy. Interpretation: Women who were exposed to DES in utero tend to reach natural menopause at an earlier age than unexposed women. The causal mechanism for this association is unclear but may be related to a reduction in the size of the original follicle pool, more rapid depletion of follicles, or changes in steroid hormone synthesis and metabolism. Age at natural menopause has been previously related to several exposures, including smoking and parity, but this study is the first to suggest that an in utero exposure may affect reproductive lifespan.

Amplification of Sense-Stranded Prokaryotic RNA

Microarray expression analysis has proven to be a valuable methodology. In eukaryotic systems where RNA is limiting, established protocols for amplification of mRNA, which rely on the poly(A) tails, are well established. In contrast, the difficulty in amplifying prokaryotic mRNA has limited the application of microarrays to microbiology. Here we present a method for the Linear Amplification of Prokaryotic Transcripts (LAPT) that is efficient and unbiased. The overhang tailing activity of Moloney murine leukemia virus reverse transcriptase is used to add the T7 promoter to cDNAs during reverse transcription. The promoter addition is uncoupled from the initial priming event allowing the promoter to be attached to the 5' end of the RNA transcript. This enables the amplification of sense-stranded RNA that is representative of the complexity and distribution of the original transcript pool. In microarray assays amplified prokaryotic RNA (10 ng total RNA starting material) showed good Spearman correlations to an unamplified control sample. Using genome-directed primers to bias addition of a T7-promoter to bacterial transcripts allowed amplification of prokaryotic transcripts in the presence of mammalian RNA (at a eukaryotic/prokaryotic RNA ratio of 500 to 1). This technology should facilitate the study of prokaryotic transcriptomes in situations, such as in vivo studies or mixed microbial populations, where the prokaryotic RNA amount is limited and/or the nontarget/target RNA ratios is high.

P-706: Do late maturing oocytes enhance the ICSI outcome?

To optimize the chances of normal fertilization, MI stage oocytes at time of cumulus cell removal are reassessed within a few hours of in vitro incubation for PB extrusion, following which they are injected. While such oocytes exhibiting this delay in maturation are generally considered as equivalent to the original MII pool, specific data on the outcome of their use is limited.

Isolation and Characterization of Low Sulfated Heparan Sulfate Sequences with Affinity for Lipoprotein Lipase

Lipoprotein lipase (LPL), which is an important enzyme in lipid metabolism, binds to heparan sulfate (HS) proteoglycans. This interaction is crucial for several aspects of LPL function, such as intracellular/extracellular transport and high capacity attachment to cell surfaces. Retention of LPL on the capillary walls, and elsewhere, via HS chains is most likely affected by the quality and quantity of HS present. Earlier studies have demonstrated that LPL interacts with highly sulfated HS and heparin oligosaccharides. Since such structures are relatively rare in endothelial HS, we have re-addressed the question of physiological ligand structures for LPL by affinity purification of end-labeled oligosaccharides originating from heparin and HS on immobilized LPL. By a combination of chemical modification and fragmentation of the bound material we identified that the bound fraction contained modestly sulfated oligosaccharides with an average sulfation of one O-sulfate per disaccharide unit and tolerates N-acetylated glucosamine residues. Therefore LPL, containing several clusters of positive charges on each subunit, may constitute an ideal structure for a protein that needs to bind with reasonable affinity to a variety of modestly sulfated sequences of the type that is abundant in HS chains.

Rolling circle amplification-RACE: a method for simultaneous isolation of 5′ and 3′ cDNA ends from amplified cDNA templates

Isolation of full-length gene transcripts is important to determine the protein coding region and study gene structure. However, isolation of novel gene sequences is often limited to expressed sequence tags (ESTs) (i.e., short cDNA fragments that predominantly represent the 3′ end of the transcript). Rapid amplification of cDNA ends (RACE) is today by far the most popular approach for obtaining full-length cDNAs when only part of the transcript’s sequence is known.Since its original description (1,2) numerous modifications and improve-ments of the method have been developed and consist of a collection of PCR-based cloning procedures that extend a known cDNA fragment toward the 3′ (3′ RACE) or the 5′ (5′ RACE) cDNA end. The original method is based on attachment of an anchor sequence to one end of the cDNA that can be used as a primer binding template in PCR with a second gene-specific primer from the known part of the gene. Although this procedure seems in theory fast and simple, it is technically difficult and usually requires substantial optimi-zation and several repetitions before satisfactory results can be obtained (3). This is particularly due to the use of a universal primer corresponding to the anchor sequence present in all cDNAs, which may result in a high background of nonspecific products even after a nested PCR with a gene-specific primer internal to the first gene-specific primer is performed. Another drawback of the method is the difficulty of obtaining the full-length 5′ end of the transcript due to the presence of many truncated transcripts in the messenger RNA (mRNA) pool. Several strategies aimed at eliminating these problems have been developed (4–9) and have proven to be very useful in certain applica-tions. One improvement is based on the utilization of a pair of gene-specific primers in inverse PCR on circularized cDNA templates, which would avoid the use of a universal primer and the problems it may generate (4,6,8–10). This strategy also allows the simulta-neous isolation of both cDNA ends in a single reaction (9,10). Some of these procedures require the generation of double-stranded cDNA, including the use of template-switching reverse transcription (9) or a post-reverse transcription adaptor ligation step (10). Methods that are performed directly on first-strand cDNA are complicated by the low efficiency of RNA ligase for the circularization reaction (6) or the need for bridging oligonucleotides for this step (8). Furthermore, existing inverse-RACE methods typically require nested PCR to amplify the transcript of interest, and only a limited number of transcripts can be isolated from a single reverse transcription reaction, making it difficult to analyze rare transcripts from scarce tissue.We describe here an improved inverse-RACE method, which uses CircLigase™ (Epicentre Biotechnologies, Madison, WI, USA) for cDNA circularization, followed by rolling circle amplification (RCA) of the circular cDNA with ϕ29 DNA polymerase (New England Biolabs, Ipswich, MA, USA). In this way, a large amount of the PCR template is produced, allowing the simultaneous isolation of the 3′ and 5′ unknown ends of a virtually unlimited number of transcripts after a single reverse transcription reaction. Figure 1 illus-trates this method, named RCA-RACE. The process takes advantage of the properties of CircLigase to circularize