Analysis of Alu Density and Gene Density

corresponding chromosomal positions) are referred to as the Human July 2003-hg16 assembly, available at the UCSC Web site. The detailed analysis of repetitive element com- position of the genomic sequence corresponding to the QM376-400 DNA fragment and to the surrounding por- tion of the YTHDF2 fourth intron (chr. 1: 28,773,429– 28,775,861) was performed using the Censor program (27) and the Repbase database of repetitive elements (28). The Alu density in a 2.6 Mb genomic region including the YTHDF2 gene (chr. 1: 27,500,001–30,100,000) was calcu- lated in 100 kb intervals by means of the Repeat Masker program (http://www.repeatmasker.org). Gene density was

Isolation of the Inter-Alu PCR Product, Cloning, and Sequencing Analysis

A genomic DNA sample was used as a template for an inter-Alu PCR, performed using the same conditions

from 16 human adult tissues (MTC Panel I and MTC Panel II) were analyzed using the conditions previously described for YTHDF2 and ACTB real-time PCR assay; the assay for a second housekeeping gene (GAPD) was added for a more reliable normalization of the results. Primers for GAPD were 59-TGAAGGTCGGAGTCAACGGATTTGGT-39 and 59- CATGTGGGCCATGAGGTCCACCAC-39 (BD Biosciences Clontech); amplification conditions were: primers 0.044 lM; cycles 958C for 30 seconds, 668C for 30 seconds, and 688C for 110 seconds. The linearity and the efficiency of the PCR amplification obtained with each primer set were checked in each assay, analyzing standard curves generated using increasing amounts of a standard cDNA (a mix of different samples) and plotting Ct values versus logarithms of cDNA concentration.

calculated in a 1.6 Mb region (chr. 1: 27,600,00–29,200,000) based on the ‘‘RefSeq Genes track’’ (29), available at the UCSC Web site. To evaluate the Alu density in the whole genome, data about the position and the length of all the Alu sequences identified in the human genome were down- loaded from the UCSC Web site (Repeat Masker analysis on the Jul 2003 assembly) and analyzed. The Alu density was calculated in nonoverlapping 1 Mb windows on each chromosome. Intervals including more than 0.4 Mb of sequencing gaps were not considered in the analysis.

Primers for the specific amplification of the YTHDF2 intron 4 microsatellite polymorphic locus were YTHDF2 I.F. (59-GAAGGACCTATCAGAGGCAGTTTT-39) and YTHDF2 I.R. (59-TTGCAGTGAGCCAAGATA-39), de- signed using Primer3 software (30). The first primer was labeled with HEX (hexachlorofluorescein) fluorochrome. PCR reagents and buffers were: 10 pmol each primer, 200 lM each deoxynucleotide, 50 mM KCl, 10 mM Tris– HCl pH 8.3, 1.5 mM Mg2þ, 1 unit of Taq DNA polymerase (Eppendorf). PCR was performed on an Applied Biosystems 9700 thermal cycler, with predenaturation at 948C for 4 minutes and 33 amplification cycles as follows: 948C for 45 seconds; 608C for 45 seconds; 728C for 40 seconds; and final extension at 728C for 7 minutes. PCR products were separated and analyzed by capillary electrophoresis on an ABI/Prism 310 Genetic Analyzer (Applied Biosystems). Six PCR fragments resulting from the amplification of different genomic DNA samples were sequenced, using the same protocols and materials reported above. YTHDF2 I.F. and YTHDF2 I.R. were respectively used as primers for sequencing reactions.

A comparative Ct (threshold cycle) method was used to determine gene expression (31,32). In the real-time PCR assay on cDNA obtained from immortalized lymphocytes, the relative abundance of YTHDF2 cDNA versus the cDNA of the housekeeping gene ACTB was expressed as [(cDNA)YTHDF2/(cDNA)ACTB] ratio of each sample, normalized by the [(cDNA)YTHDF2/(cDNA)ACTB] ratio of a control sample (calibrator). The normalized ratio was calculated taking into account the different PCR efficiencies for target and reference amplification (32). For cDNA samples from human tissues (MTC Panels I and II), two housekeeping genes were used: ACTB and GAPD. For each tissue obtained, two different values for YTHDF2 expres- sion were normalized against ACTB or GAPD, respectively: YTHDF2 expression normalized against that of the two housekeeping genes was given by the geometric mean of the two values in each sample (33). Intra-assay and inter-assay coefficients of variation were 16.1% and 24.4%, calculated repeating some samples in the same assay and in different assays, respectively.

Reverse Transcription and Real-Time PCR

The total RNA of immortalized lymphocytes derived from 51 participants (35 centenarians and 16 young individ- uals) was reverse transcribed with M-MLV reverse tran- scriptase (Ambion, Austin, TX). The cDNA was analyzed using a semiquantitative real-time PCR to assess the expres- sion of the YTHDF2 gene relative to the housekeeping gene ACTB. PCR was performed on the iCycler (Bio-Rad, Hercules, CA), using SYBR Green (Bio-Rad) as a fluores- cent dye for the real-time detection. Primers for YTHDF2 cDNA were HR1 (59-ACCTTACTTGAGTCCACAGG-39) and HR2 (59-AGCCAATGGAGGGACTGTAG-39), respec- tively complementary to sequences situated in the second and third exon of the gene, and designed using Primer3 software (30). Primers for b-actin reverse transcription (RT)–PCR were F.B.Act. (59-GCGAGAAGATGACCCA- GATC-39) and R.B.Act. (59-GGATAGCACAGCCTGGA- TAG-39). PCR mixes for both RT–PCRs had a final volume of 20 ll, and were composed of 1 X buffer Sybr Green Supermix (Bio-Rad), primers (0.6 lM HR1 and HR2 for YTHDF2 RT–PCR; 0.15 lM F.B.Act. and R.B.Act. for beta-actin RT–PCR), and 2 ll of cDNA (derived from 40 ng of total RNA) from immortalized lymphocytes. Each sample was assayed in duplicate. The specificity of the PCR ampli- fication was checked with a heat dissociation protocol after the final cycle. Two nanograms of cDNA samples derived

Statistical Analysis