Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry

Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.     

Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ)

We assess the reliability of isobaric-tags for relative and absolute quantitation (iTRAQ), based on different types of replicate analyses taking into account technical, experimental, and biological variations. In total, 10 iTRAQ experiments were analyzed across three domains of life involving Saccharomyces cerevisiae KAY446, Sulfolobus solfataricus P2, and Synechocystis sp. PCC 6803. The coverage of protein expression of iTRAQ analysis increases as the variation tolerance increases. In brief, a cutoff point at +/-50% variation (+/-0.50) would yield 88% coverage in quantification based on an analysis of biological replicates. Technical replicate analysis produces a higher coverage level of 95% at a lower cutoff point of +/-30% variation. Experimental or iTRAQ variations exhibit similar behavior as biological variations, which suggest that most of the measurable deviations come from biological variations. These findings underline the importance of replicate analysis as a validation tool and benchmarking technique in protein expression analysis.     

Enumeration of CD4(+) T-cells in the peripheral blood of HIV-infected patients: an interlaboratory study of the FACSCount system.

The aim of the present study was to assess the interlaboratory reproducibility of the FACSCount system for the enumeration of peripheral blood (PB) CD4(+) T-cells. In each of the seven participating centers, both previously stained and unstained PB samples (n = 49) were received and either analyzed or stained and then analyzed. Interlaboratory reproducibility was checked in two different groups of centers (n = 3 and n = 4) where the study was performed in parallel. In addition, both the intralaboratory precision and accuracy of this system were analyzed in comparison with results obtained with conventional flow cytometry. Accordingly, upon comparing both methods, a high degree of correlation was observed in the total number of CD3(+) T-cells (coefficient of correlation of 0.9750 +/- 0.0184, slope of the best linear fit: 0. 9214 +/- 0.0311, y-intercept of 12 +/- 47) as well as in the number of CD3(+)/CD4(+) (coefficient of correlation of 0.9794 +/- 0.1457, slope of the best linear fit: 0.9463 +/- 0.0753, y-intercept of -11 +/- 36) and CD3(+)/CD8(+) (coefficient of correlation of 0.9728 +/- 0.0192, slope of the best linear fit: 0.9682 +/- 0.0735, y-intercept of 7 +/- 95) major subsets. In addition, low coefficients of variation (CV) were obtained for replicates, indicating the method's high degree of accuracy. The present study shows that with respect to the interlaboratory reproducibility reported for most techniques used for the enumeration of PB CD4(+) T-cells, the FACSCount system results in data with much lower coefficients of variance (CVs) (mean CV of less than 10%). Upon measuring the impact on results of different variables associated with either sample preparation or data acquisition and analysis, our study clearly shows that data acquisition and analysis does not influence the results by increasing variability since the coefficients of variation obtained for samples prepared in the same laboratory under the same conditions and read in different laboratories with different instruments were identical to those obtained for the replicates of the same samples read in each individual center. In contrast, interlaboratory variability, although low, significantly increased when sample preparation was carried out in different laboratories, suggesting that pipetting still represents the major source of variability in the FACSCount system.     

Application of microfluidic devices to proteomics research: identification of trace-level protein digests and affinity capture of target peptides

This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 microl total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C(18) reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of approximately 5 nM (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2-1.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gel-isolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system.     

The influence of sample preparation and replicate analyses on HeLa Cell phosphoproteome coverage

Ongoing optimization of proteomic methodologies seeks to improve both the coverage and confidence of protein identifications. The optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample), and biological replicates (multiple individual samples) are crucial in proteomic studies to avoid the pitfalls associated with single point analysis and under-sampling. Phosphopeptides were isolated from HeLa cells and analyzed by nano-reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (nano-RP-LC-MS/MS). We observed that a detergent-based protein extraction approach, followed with additional steps for nucleic acid removal, provided a simple alternative to the broadly used Trizol extraction. The evaluation of four technical replicates demonstrated measurement reproducibility with low percent variance in peptide responses at approximately 3%, where additional peptide identifications were made with each added technical replicate. The inclusion of six technical replicates for moderately complex protein extracts (approximately 4000 uniquely identified peptides per data set) affords the optimal collection of peptide information.     

Revisiting the effect of PCR replication and sequencing depth on biodiversity metrics in environmental DNA metabarcoding

Environmental DNA (eDNA) metabarcoding is an increasingly popular tool for measuring and cataloguing biodiversity. Because the environments and substrates in which DNA is preserved differ considerably, eDNA research often requires bespoke approaches to generating eDNA data. Here, we explore how two experimental choices in eDNA study design—the number of PCR replicates and the depth of sequencing of PCR replicates—influence the composition and consistency of taxa recovered from eDNA extracts. We perform 24 PCR replicates from each of six soil samples using two of the most common metabarcodes for Fungi and Viridiplantae (ITS1 and ITS2), and sequence each replicate to an average depth of ~84,000 reads. We find that PCR replicates are broadly consistent in composition and relative abundance of dominant taxa, but that low abundance taxa are often unique to one or a few PCR replicates. Taxa observed in one out of 24 PCR replicates make up 21–29% of the total taxa detected. We also observe that sequencing depth or rarefaction influences alpha diversity and beta diversity estimates. Read sampling depth influences local contribution to beta diversity, placement in ordinations, and beta dispersion in ordinations. Our results suggest that, because common taxa drive some alpha diversity estimates, few PCR replicates and low read sampling depths may be sufficient for many biological applications of eDNA metabarcoding. However, because rare taxa are recovered stochastically, eDNA metabarcoding may never fully recover the true amplifiable alpha diversity in an eDNA extract. Rare taxa drive PCR replicate outliers of alpha and beta diversity and lead to dispersion differences at different read sampling depths. We conclude that researchers should consider the complexity and unevenness of a community when choosing analytical approaches, read sampling depths, and filtering thresholds to arrive at stable estimates.     

Label-free detection of differential protein expression by LC/MALDI mass spectrometry

Protein abundance changes during disease or experimental perturbation are increasingly analyzed by label-free LC/MS approaches. Here we demonstrate the use of LC/MALDI MS for label-free detection of protein expression differences using Escherichia coli cultures grown on arabinose, fructose or glucose as a carbon source. The advantages of MALDI, such as detection of only singly charged ions, and MALDI plate archiving to facilitate retrospective MS/MS data collection are illustrated. MALDI spectra from RP chromatography of tryptic digests of the E. coli lysates were aligned and quantitated using the Rosetta Elucidator system. Approximately 5000 peptide signals were detected in all LC/MALDI runs spanning over 3 orders of magnitude of signal intensity. The average coefficients of variation for all signals across the entire intensity range in all technical replicates were found to be <25%. Pearson correlation coefficients from 0.93 to 0.98 for pairwise comparisons illustrate high replicate reproducibility. Expression differences determined by Analysis of Variance highlighted over 500 isotope clusters ( p < 0.01), which represented candidates for targeted peptide identification using MS/MS. Biologically interpretable protein identifications that could be derived underpin the general utility of this label-free LC/MALDI strategy.     

Whole cell,label free protein quantitation with data independent acquisition: Quantitation at the MS2 level

Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition—SWATH—to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (～40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R² ～ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R² ～ 0.930), indicating that this approach may have increased viability compared to recent applications in label free protein quantitation. SWATH based quantitation was able to consistently detect differences in relative protein quantity and it provided coverage for a number of proteins that were missed in some samples by DDA analysis.     

Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens

RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ～10 000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction.     

Total and high-density lipoprotein cholesterol measurements. Hazards in clinical interpretation.

Duplicate plasma specimens from 24 persons were sent to a community hospital, a commercial laboratory, and a university lipid research laboratory at two separate times to assess the intralaboratory and interlaboratory variations of total and high-density cholesterol measurements. For all three laboratories, the 95% confidence limits for the reproducibility of total cholesterol levels are about +/- 5.5%. For high-density lipoprotein (HDL) values, they are +/- 7%, +/- 14.45%, and +/- 9%. Interlaboratory differences for total cholesterol at the 95% confidence level are +/- 7.7% and for HDL +/- 18.4%. Construction of a "cardiac risk ratio" of total to HDL cholesterol levels is subject to confusion because small errors in HDL cholesterol levels produce large errors in the ratio.     

Plasma desorption mapping of the tryptic digest of 23-kDa recombinant human growth hormone

Plasma desorption mass spectrometry has been used to map the tryptic fragments from the 23-kDa recombinant human growth hormone protein. The unfractionated tryptic digest was adsorbed directly onto a nitrocellulose sample foil and mass spectra were obtained in both the positive and the negative ion mode. The adsorbed sample was then washed with deionized water and its mass spectrum was again obtained. The latter spectrum revealed tryptic fragments that were not observed in the spectra of the unwashed sample, which can be attributed (to some extent) to the removal of hydrophilic residues during washing. From this study a protocol, aimed at the complete mapping of tryptic fragments, is outlined.     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

Differential recovery of peptides from sample tubes and the reproducibility of quantitative proteomic data

Differential recovery of peptides due to nonspecific adsorption can seriously compromise reproducibility and quality of proteomic data for peptide analyses by liquid chromatography-mass spectrometry (LC-MS). This study demonstrates large variations in reproducibility and quantitation of LC-MS data for peptides derived from tryptic digests of BSA upon storage in different sample tubes. Notably, we show that highly improved consistency and lower errors in quantitation of BSA tryptic peptides in replicate measurements is achieved with low-retention tubes compared to regular eppendorf tubes. Furthermore, qualitative differences in peptides detected by LC-MS were observed in the two types of storage tubes. These results illustrate the necessity for careful evaluation of storage vessels and conditions to minimize variability in sample quality for LC-MS experiments.     

High-performance tryptic mapping of recombinant bovine somatotropin

Experiments are described that have lead to the development of a highly reproducible tryptic map of recombinant DNA derived bovine somatotropin (rbSt). Tryptic digestion of rbSt at 37 degrees C results in the formation of a precipitate. Preliminary characterization of the precipitate suggests that its formation is due to the association of intermediate tryptic fragments. An examination of the temperature dependence of the digestion has revealed that precipitate formation is inhibited when digestion is performed at 10 degrees C or less. The combination of a 5-mg sample, the use of highly purified trypsin, and digestion at 5 degrees C generate a tryptic map that exhibits an average 1.3% RSD (0.5-3.6%) for all anticipated fragments. Validation studies demonstrate that while the peak response precision is rugged to daily variation of operators or chromatographic systems, the fragment retention is not. This dictates that peaks be assigned by qualitative pattern recognition. Assay ruggedness in the peak response domain allows for the implementation of quantitative methods for the comparison of rbSt reference standard and sample tryptic maps. The assay is linear for all anticipated fragments within 50-150% of the operating range. Specificity is established by assay of pituitary somatotropins from other species and rbSt analogs produced by site-specific mutagenesis. The data demonstrate that all single amino acid substitutions examined are identified by using the technique. Assay sensitivity is validated for selected tryptic fragments through analysis of reference standard digests spiked with known amounts of rbSt analog digests. The data indicate that potential impurities of 3.2, 2.0, and 4.5% can be quantitated with statistical confidence in the tryptic fragments T1, T10, and T23 + 25, respectively.     

Comparison of label-free methods for quantifying human proteins by shotgun proteomics

Measurements of mass spectral peak intensities and spectral counts are promising methods for quantifying protein abundance changes in shotgun proteomic analyses. We describe Serac, software developed to evaluate the ability of each method to quantify relative changes in protein abundance. Dynamic range and linearity using a three-dimensional ion trap were tested using standard proteins spiked into a complex sample. Linearity and good agreement between observed versus expected protein ratios were obtained after normalization and background subtraction of peak area intensity measurements and correction of spectral counts to eliminate discontinuity in ratio estimates. Peak intensity values useful for protein quantitation ranged from 10(7) to 10(11) counts with no obvious saturation effect, and proteins in replicate samples showed variations of less than 2-fold within the 95% range (+/-2sigma) when >or=3 peptides/protein were shared between samples. Protein ratios were determined with high confidence from spectral counts when maximum spectral counts were >or=4 spectra/protein, and replicates showed equivalent measurements well within 95% confidence limits. In further tests, complex samples were separated by gel exclusion chromatography, quantifying changes in protein abundance between different fractions. Linear behavior of peak area intensity measurements was obtained for peptides from proteins in different fractions. Protein ratios determined by spectral counting agreed well with those determined from peak area intensity measurements, and both agreed with independent measurements based on gel staining intensities. Overall spectral counting proved to be a more sensitive method for detecting proteins that undergo changes in abundance, whereas peak area intensity measurements yielded more accurate estimates of protein ratios. Finally these methods were used to analyze differential changes in protein expression in human erythroleukemia K562 cells stimulated under conditions that promote cell differentiation by mitogen-activated protein kinase pathway activation. Protein changes identified with p<0.1 showed good correlations with parallel measurements of changes in mRNA expression.     

In vitro development of yak (Bos grunniens) embryos generated by interspecies nuclear transfer

Interspecies cloning may be used as an effective method to conserve highly endangered species, but at present it suffers from relatively low levels of efficiency. In order to find a technique that could be used in conservation of the wild yak (Bos grunniens), we designed in six separate experiments to investigate the following factors that might influence the efficiency of interspecies cloning: (1) maturation rates of the recipient bovine oocytes; (2) nuclear donor cell types; (3) age of the yak from which the yak ear skin fibroblast cell line originated; (4) donor cells treated with or without serum starvation; (5) nuclear donor gained from fresh cells or frozen-thawed cells; (6) effect of 0.5 or 1.5 h from fusion to activation. The results of experiment 1 showed that when recipient oocytes in a replicate had a maturation rate of <40% (34+/-3.0%; three replicates) the proportion of nuclear transferred oocytes that developed to blastocyst was 2+/-1.1%, which was significantly lower (P<0.01) than the 25+/-3.2% achieved when the recipient oocyte maturation rate was 71+/-3.7% (three replicates). The efficiency of blastocyst production was increased substantially (P<0.05) when the time from fusion to activation increased from 0.5 h (21+/-2.3%; three replicates) to 1.5 h (35+/-3.5%; five replicates; experiment 6). There was no significant effect of the source of the donor nuclei (ear skin fibroblast or cumulus cells), the age of the animal (3 months or 4 years) from which the donor cells were derived, serum deprivation of the donor cells, or the use of fresh or frozen-thawed donor cells (experiments 2-5). Transfer of three interspecies cloned blastocysts to each of 108 bovine recipients resulted in two pregnancies being established that did not survive to day 120 of gestation.     

Effects of 2 wk of GH administration on 24-h indirect calorimetry in young, healthy, lean men

The present study was designed as a randomized, double-blind placebo (Plc)-controlled study to determine the effect of 2 wk of growth hormone administration (GH-adm.) on energy expenditure (EE) and substrate oxidation in healthy humans. Sixteen young healthy men were divided into two groups. The study consisted of two 24-h measurements (indirect calorimetry), separated by 2 wk of either Plc or GH injections (6 IU/day). At baseline, no significant differences were observed between the two groups in any of the measured anthropometric, hormonal, or metabolic parameters, neither did the parameters change over time in the Plc group. GH-adm. resulted in a 4.4% increase in 24-h EE (P < 0.05) and an increase in fat oxidation by 29% (P < 0.05). However, a decrease in the respiratory quotient was only observed in the postabsorptive phase after an overnight fast (0.84 +/- 0.1 to 0.79 +/- 0.1, P < 0.05). Furthermore, lean body mass (LBM) was increased by GH-adm. only [62.8 +/- 2.5 kg (baseline) vs. 64.7 +/- 2.4 kg (after), P < 0.001]. In conclusion, GH-adm. increases 24-h EE, which may be partly explained by increased LBM. Furthermore, GH-adm. stimulates fat combustion, especially in the postabsorptive state.     

Impact of scoring error and reproducibility RAPD data on RAPD based estimates of genetic distance

RAPD band reproducibility and scoring error were evaluated for RAPDs generated by 50 RAPD primers among ten snap bean (Phaseolus vulgaris L.) genotypes. Genetic distances based on different sets of RAPD bands were compared to evaluate the impact of scoring error, reproducibility, and differences in relative amplification strength on the reproducibility of RAPD based genetic distance estimates. The measured RAPD data scoring error was 2%. Reproducibility, expressed as the percentage of RAPD bands scored that are also scored in replicate data, was 76%. The results indicate that the probability of a scored RAPD band being scored in replicate data is strongly dependent on the uniformity of amplification conditions between experiments, as well as the relative amplification strength of the RAPD band. Significant improvement in the reproducibility of scored bands and some reduction in scoring error was achieved by reducing differences in reaction conditions between replicates. Observed primer variability for the reproducibility of scored RAPDs may also facilitate the selection of primers, resulting in dramatic improvements in the reproducibility of RAPD data used in germplasm studies. Variance of genetic distances across replicates due to sampling error was found to be more than six times greater than that due to scoring error for a set of 192 RAPD bands. Genetic distance matrices computed from the RAPD bands scored in replicated data and RAPD bands that failed to be scored in replicated data were not significantly different. Differences in the ethidium bromide staining intensity of RAPD bands were not associated with significant differences in resulting genetic distance matrices. The assumption of sampling error as the only source of error was sufficient to account for the observed variation in genetic distance estimates across independent sets of RAPD bands.     

Spearman's footrule as a measure of cDNA microarray reproducibility

Replication is a crucial aspect of microarray experiments, due to various sources of errors that persist even after systematic effects are removed. It has been confirmed that replication in microarray studies is not equivalent to duplication, and hence it is not a waste of scientific resources. Replication and reproducibility are the most important issues for microarray application in genomics. However, little attention has been paid to the assessment of reproducibility among replicates. Here we develop, using Spearman's footrule, a new measure of the reproducibility of cDNA microarrays, which is based on how consistently a gene's relative rank is maintained in two replicates. The reproducibility measure, termed index.R, has an R2-type operational interpretation. Index.R assesses reproducibility at the initial stage of the microarray data analysis even before normalization is done. We first define three layers of replicates, biological, technical, and hybridizational, which refer to different biological units, different mRNAs from the same tissue, and separate cDNAs from a cDNA pool. As the replicate layer moves down to a lower level, the experiment has fewer sources of errors and thus is expected to be more reproducible. To validate the method we apply index.R to two sets of controlled cDNA microarray experiments, each of which has two or three layers of replicates. Index.R shows a uniform increase as the layer of the replicates moves into a more homogeneous environment. We also note that index.R has a larger jump size than Pearson's correlation or Spearman's rank correlation for each replicate layer move, and therefore, it has greater expandability as a measure in [0,1] than these two other measures.