186 Age-related molecular variability in Caenorhabditis elegans, T. R. Golden et al. Chromosome V = her-1 (ZK287.8). Five primer sets were gen- erated, as follows: I.F tcgctcgatgatgaatcttg I.R cctgtggtcctggtcctcta II.F caagggaccgaaatctcgta II.R cagagtgagtcgtgaaccga III.F aagcagcaggactcacgaat III.R tgcacatgtcgttatggctt IV.F atccaatacaggtcgatggc IV.R cagctcctcctggatgtttc V.F ttatgacagggatccgaagc V.R aaaaacgctcaccatccaag Primer pairs were optimized by performing PCR on a serial dilution of bulk worm extract in order to determine primer efficiency, resulting in the elimination of primer pairs for chromosomes I and V and the validation of primer pairs for chro- mosomes II, III, and IV with efficiencies not significantly different from 100%. These primer pairs were used in all subsequent studies. PCR conditions PCR reactions (10 µ L per well) contained 5 µ L worm extract (equivalent to 1/30 of an extract from a single worm), 500 nM forward and reverse primers, and 1× SYBR Master Mix (Applied Biosystems, Foster City, CA, USA). Reactions were set up in a larger volume (2.5× volume = 25 µ L total) and aliquoted into duplicate 10 µ L wells for real-time amplification. Cycling con- ditions were: 95 °C × 10 min to activate hot start Taq polymerase followed by 40 cycles of 95 °C × 15 s + 60 °C × 1 min, followed by a dissociation (melt) stage ramping from 60 °C to 95 °C at 2% of maximal ramp rate. Cycling was achieved in 384 well plates on a 7900 HT Sequence Detection System (Applied Biosystems), and fluorescence intensity data was collected at the annealing stage (60 °C) and during dissociation. Raw data were converted to cycle threshold (Ct) values using SDS version 2.1.1 software (Applied Biosystems), and relative quantities calculated using the delta Ct method. For example, for the N2 hermaph- rodites, there were a total of 9 time points × 15 worms per time point = 135 samples × 2 wells per sample = 270 wells per chromosome primer pair, that were run on a single 384 well PCR plate. Hence for these samples, a total of three 384 well plates were run, one for each primer pair. Standards and quantitation All extracts for a given strain were run on a single 384 well PCR plate, with the exception of daf-2, which was split onto two plates (as there were a total of 13 × 15 × 2 = 390 wells required). In order to ensure that there was no plate-to-plate variation in PCR amplification efficiency, PCR run plates contained 22 replicate wells of a master extract of a pool of N2 worms, for comparison across plates run at different times. There were no significant differences between plates run using a given primer pair in the mean Ct of these positive controls. All plates also contained no-template control wells, none of which amplified. Melt curve analysis was used to ensure that amplified wells contained actual amplicons, as opposed to primer dimers (which were very rare). Conversion of Ct values into copy number was performed using dsDNA standards (PCR amplicons, purified and quantitated using Picogreen dye and the calculated molecular weight for the length of the dsDNA product). From each strain assayed, 15 samples (calibrators) from the youngest age animals were amplified together a second time on a plate containing a standard curve of known copy number of dsDNA standards. Cycle thresholds from these runs were used to calculate copy numbers for all 15 calibrators, which were then used to convert prior Ct values into copy numbers. These quan- tifications were performed independently for each set of primer pairs. Statistical analysis To compare mean nuclear genome content between two groups of individuals, we carried out an unpaired t-test. We tested the trend in variance with age by simply (i) calcu- lating the sample variance at each age, and (ii) regressing this sample variance vs. age. In order to derive robust inference, we used a permutation test (using the typical Z-statistic on the slope of each regression) to derive exact P values. For testing the equivalence of a variance at a set of time point (i.e., between two different strains), we used a variance ratio test (Pagano & Gauvreau, 1993). DAPI staining Nematodes were fixed overnight in Carnoy’s solution, allowed to dry on slides overnight, and stained with mounting medium containing DAPI. Imaging was conducted on a Zeiss Axioplan 2 microscope. We collected low-magnifcation (10×) images of each worm, and high magnification (63×) optical sections of the middle and the tail of each worm. Images were deconvoluted using deconvolution software from either Huygens scientific or AutoDeblur (AutoQuant). Both deconvolution algorithms significantly improved the images over the raw images acquired, whereas maintaining image detail. After deconvolution, tail image stacks were assembled into a three-dimensional image using a combination of Autodeblur and Aftereffects (Adobe). BrdU labeling N2 nematodes at 7, 9, and 11 days of age were exposed to BrdU by adding 0.5 mL of a 10-mM solution of BrdU in S medium to 6 cm plates containing the worms. The worms were left on the plates overnight, collected, rinsed, and fixed in 4% paraformaldehyde. After fixation, worms were permeabilized by exposure to β-mercaptoethanol followed by collagenase digestion of the cuticle. Worms were then exposed to 2N HCl for 1 h. This acid treatment was found to be necessary to allow the anti-BrdU antibody access to incorporated BrdU. However, this treatment interfered with the ability to detect DNA with DAPI or other DNA-reactive dyes, so we were unable to collect images of both DAPI-stained and BrdU-labeled DNA from the same worm. BrdU was detected with a primary monoclonal antibody (Sigma, St. Louis, MO, USA), and a fluorescent labeled goat antimouse secondary antibody (Molecular probes, Eugene, OR, USA). © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007 No claim to original US government works