Diets

The non-supplemented CRM diet was supplied by Dietex International, Witham, UK. The LA-supplemented diet was the CRM diet enriched with a racemic mixture of DL-thioctic acid (α-lipoic acid) obtained from Fisher Scientific UK Ltd, at 1.5 g/kg by the Special Diet Systems Division of Dietex International. LA is known to have an anti-obesity effect by inhibiting food intake and retarding growth [66]; therefore, in the previous study by Merry et al. [10], it was ensured that the dose supplied to the animals did not inhibit the growth rate to induce an indirect dietary restriction effect.

Animals

A previous experiment supplied the rat brain tissue for this study [10]. All animal husbandry procedures undertaken in this study were carried out in accordance with the provisions of the United Kingdom Animals (Scientific Procedures) Act 1986. The animal study from which the tissue was generated for the current publication was conducted under the Home Office Project Licence 40/2964, held by B.J.M. The work was also reviewed by the University of Liverpool Ethical Committee for Animal Welfare. Male BN rats (SubstrainBN/SsNOlaHSD) were obtained from Harlan UK at 21–28 days of age and maintained under barrier conditions on a 12-h light:12-h dark cycle (08:00–20:00). The health status of the rats was monitored at regular intervals through the screening of sentinel animals. All animals were caged in groups of four and fed the non-supplemented CRM diet AL until 2 months of age, when they were transferred to single housing and assigned randomly to one of six dietary groups as summarised in Table 1. All rats fed AL and CR were sacrificed at 6, 12, and 28 months of age. Animals subjected to CR were fed either the CRM diet or the LA-supplemented CRM diet at 55 % the daily food intake of control rats to maintain their body weight at approximately 55 % that of age matched AL-fed animals. The diet of animals maintained on a CR regime was supplied daily as pre-weighed rations between 10:30 and 11:00 h. The daily food ration supplied to CR animals was the same for age-matched animals irrespective of whether they were fed the CRM diet or the LA-supplemented CRM diet. Six dietary feeding combinations were studied to compare the effect of AL or CR feeding with and without LA supplementation. Survival trajectories for these groups were previously determined [10]. All groups were run simultaneously and so experienced identical husbandry and housing conditions. None of the animals exhibited any signs of pathology when sacrificed. Each age group had six rats from which tissue samples were taken, flash frozen and stored at −80 °C.

Cortex dissection and RNA and miRNA extraction

To minimise thawing and therefore degradation of RNA, the cerebral cortex was removed from the whole brain on a solid CO 2 base under a dissecting microscope. The cerebral cortex was cut into small pieces to aid RNA extraction.

RNA was extracted from the cerebral cortex using Qiagen's TissueLyser II and RNeasy lipid tissue kit. The quality of the extracted RNA was assessed using the Agilent 2100 Bioanalyser; all RNA integrity numbers (RINs) were above 8, indicating that good quality RNA had been extracted. Each sample was split in two for either whole transcriptome or small RNA analysis. For the whole transcriptome analysis the samples were pooled two by two (leaving three samples per age/diet group). Ribosomal RNA was removed from the pooled samples using a Eukaryote ribominus kit (Invitrogen) and confirmed with the Agilent 2100 Bioanalyser. Ribosomal removal, rather than poly(A) selection, allows certain non-coding RNAs without poly(A) tails to be included in the sequencing.

The miRNA fraction was enriched using the miRVana kit. The enriched miRNA samples were then pooled as with the whole transcriptome samples.

cDNA library preparation and SOLiD sequencing

The whole transciptome library preparation protocol was carried out according to the manufacturer’s instructions. The RNA was fragmented and cleaned up using spin columns (Invitrogen) and SOLiD RNA adapters were then hybridised and ligated to the samples. Reverse transcription was performed to generate cDNA. The cDNA was purified, size selected, amplified and then purified again (as detailed in the SOLiD protocol). The size distribution of the cDNA library was assessed using the Bioanalyser. The samples were then subjected to emulsion PCR and sequenced in the Centre for Genomic Research at the University of Liverpool (https://www.liverpool.ac.uk/genomic-research/) using the SOLiD system 5500xl to generate 75-bp forward reads.

The miRNA library preparation protocol was carried out according to the manufacturer’s instructions. The samples were then subjected to emulsion PCR and sequenced in the Centre for Genomic Research at the University of Liverpool using the SOLiD system v4 to generate 50-bp forward reads.

Whole transcriptome data mapping and differential expression analysis

The RNA-seq results from the SOLiD system were output as colour space fasta and quality files, and these files were mapped to the Ensembl release 71 rat reference genome (Rnor_5.0, March 2012) using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml); the reads were filtered by quality using the –e phred quality setting (-e 400), multiple mapping reads were not allowed and the best option was used with mismatches limited to 2. For each sample approximately 36 million reads were generated. On average, 23 million reads per sample were mapped to the reference genome (approximately 63 % of reads generated were mapped; this figure is relatively low because of the conservative options specified in Bowtie but ensures that the highest quality and most robust alignments are reported). All data have been submitted to Gene Expression Omnibus (GEO) under the accession GSE57110. The differential expression analysis was carried out as in our previous study [14]. In brief, raw counts per gene were estimated by the Python script HTSeq count (http://www-huber.embl.de/users/anders/HTSeq/) and used by EdgeR [67] to estimate DE in pair-wise comparisons. Counts per million (cpm) were calculated and only genes with 1 cpm in at least three samples were included in the analysis. Trimmed mean of M-values (TMM) normalisation of the sequenced libraries was performed to remove effects due to differences in library size. EdgeR generates a FC for each gene; p values and the Benjamin-Hochberg FDR were calculated to statistically test the measured DE. As in previous studies, no effect of size cutoff was used, as ageing-related changes tend to be subtle [23].

Enrichment analysis and heatmap generation

To create heatmaps of DE genes, R and the R package heatmap3 were used along with the log2 FC output from EdgeR. To assess the biological significance of gene expression changes we used the Cytoscape plug-in ClueGO to perform an enrichment analysis on each individual comparison and then, using a built-in algorithm, the GO terms were collapsed based on related terms and statistical significance in order to give a simplified network [68, 69]. Further enrichment analysis was performed by GSEA [70, 71] and DAVID [72].

Whole transcriptome qPCR validation

To generate cDNA for qPCR, 3.5 μg of total RNA was reverse transcribed using Superscript III First-strand synthesis system for RT-PCR (Invitrogen, Paisley, UK). The Roche Universal Probe Library was used to design primers with sequences obtained from Ensembl. All primers were designed to cross an exon–exon boundary. The specificity of the primers was checked using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A reference gene experiment was conducted to identify the most stably expressed genes in the cerebral cortex with age and diet (data not shown); Hprt1 (Additional file 10), B2m (Rn00560865_m1, Applied Biosystems) and Ywhaz (Rn00755072_m1 Applied Biosystems) were the most stably expressed in all conditions. The reference genes were used to normalise the qPCR results. Additional file 10 shows the primers, amplicons, and probes used. The qPCR assays were all performed in triplicate using a TaqMan™ ABI PRISM 7500 fast (Applied Biosystems, Foster City, CA, USA) in 96-well plate format. A 20-ml reaction volume was used per well, consisting of 10 μl Taqman 2× PCR master mix (Universal PCR Mastermix, Applied Biosystems), 0.2 μl each of 20 mM forward and reverse primers, 0.2 μl of 10 mM probe (Exiqon, Roche Diagnostics Ltd), 0.2 μl distilled water and 9.2 μl of cDNA or water for the negative controls. The amplification was performed as follows: 2 min at 50 °C, 10 min at 95 °C followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The efficiency of the assays was between 93 % and 107 % and the R2 values were >0.98. The ΔΔcT method was used to measure expression. The data were further corrected by the efficiency of the standard curve for each gene. Log2 FC was calculated and compared with the RNA-seq results in order to confirm the expression results. The standard error was calculated for log2 FC as follows: (Standard error/Mean) × log2e. For qPCR the relative quantification value was used to calculate standard error. For RNA-seq, raw reads converted into relative values were used to calculate standard errors.

miRNA mapping

The RNA-seq results from the SOLiD system were output as colour space fasta and quality files. Using a custom script the base space adapter sequences was converted into colour space. A freely available Python script (cutadapt; https://cutadapt.readthedocs.org/en/stable/) was used to remove the adapter sequence and output a cfastq file. The freely available stand-alone Java program miRanalyzer [73] was used for mapping and analysis (http://bioinfo2.ugr.es/miRanalyzer/standalone.html). Using the Perl scripts provided by miRanalyzer, the reads were grouped and then mapped using Bowtie. miRanalyzer maps to a Bowtie index file for the rat; it then removes reads mapping to RefSeq (known mRNA) and Rfam (other known non-coding RNAs), leaving the user with reads that are likely to be miRNAs. New novel miRNAs are identified by miRanalyzer, detailing the secondary structure and number of times predicted by the five algorithms it uses. The results are also split into files for uniquely mapping and ambiguously mapping reads. This results in very clean results which are uniquely mapped.

miRNA differential expression analysis

The differential expression analysis was carried out using a range of custom Python scripts, conditional quantile normalisation [74] and EdgeR. Conditional quantile normalisation and Lowess normalisation have been shown to be most appropriate for miRNA analysis [75]. The known miRNAs were analysed separately from the novel predictions. Only reads with one count per million in at least three samples were included in the analysis. After normalisation and exclusion of low read counts, the differential expression analysis was carried out using EdgeR’s generalised linear model and tag-wise dispersion. A FDR cutoff of 0.05 was used.

miRNA qPCR

Validation of the miRNA differential expression results was carried out using the miScript PCR system (Qiagen). Reverse transcription using the miScript II RT kit was carried out according to the manufacturer’s instructions: 4 μl 5× miScript HiSpec buffer, 2 μl 10× miScript nucleics mix, 2 μl miScript reverse transcriptase mix into a total volume of 20 μl with 10 ng to 2 μg of template miRNA. This was incubated for 60 min at 37 °C and then 5 min at 95 °C. The resulting cDNA was diluted by adding 200 μl of distilled water. The miScript SYBR Green PCR Kit was used for the qPCR reaction. All samples were performed in triplicate in a 96-well plate format, using 2.5 μl cDNA, 12.5 μl 2× quantiTECT SYBR, 2.5 μl 10× miScript universal primers, 2.5 μl miScript primer assay, 5 μl RNase free water per well. Six small nucleolar RNAs (snoRNAs) provided and validated by Qiagen were used as the reference genes. Subsequently, a reference gene stability experiment ascertained that only three reference genes were required (Snord96a, Snord95 and Snord68).

LAG analysis

The comparison with known mouse, fly and worm LAGs was done using data from the Human Ageing Genomics Resources GenAge database, using build 16 [76]. Protein–protein interaction data for the construction of longevity networks and for the analysis of LAG partners were retrieved from the BioGRID database [77, 78], release 3.1.83. The construction of longevity networks has been described in detail previously [79]. Briefly, the networks include LAGs as a core set, and their first order interaction partners. Only the largest connected component is kept in the network.

In order to compare the rat “longevity genes” with genes from other species, orthologs were obtained using the InParanoid7 database [80]. Exclusion of inparalogs was done for the default threshold score of 0.05.

Cell culture

CTX TNA2 cells were purchased from HPA cultures (UK). The cells were maintained in DMEM (Life Tech) with high glucose, L-glutamine, phenol red, sodium pyruvate. The DMEM was supplemented with 10 % foetal bovine serum and penicillin-streptomycin-glutamine. The cells were sub-cultured three times a week. Passages 12–14 were used for the experiments.

Transfection

The confluent cells were sub-cultured and diluted 1 in 10 in antibiotic-free media and then counted using a haemocytometer. The cells were diluted to 1 × 104 and 100 μl of cell solution was added to each well of a 96-well plate. The cells were incubated overnight at 37 °C, 5 % CO 2 to allow them to attach. The transfection was performed using the Dharmacon miRNA system. Mimics and inhibitors for miR-98-3p, positive controls, including a miRNA mimic that inhibits GAPDH and a miRNA mimic that increases miR-16, and two negative controls (mimic and inhibitor) were used to assess the transfection efficiency and viability by comparing with a transfection reagent-only control and an untreated control. The viability was assessed using the Alamar Blue assay and by 96 hours the mimic was at 115 % viability compared with the transfection control, and the inhibitor was at 86 % viability compared with the transfection control (see Additional file 11 for results). The mRNA was extracted using the cells-to-CT kit (Life Tech) according to the manufacturer’s protocol. The miRNA was extracted using the miRvana kit as above. cDNA synthesis and qPCR were performed as above using Actb as the reference gene for the mRNA experiment. The transfection efficiency was ascertained by qPCR by measuring GAPDH and miR-16 knock down. GAPDH was reduced 4.2-fold and miR-16 was reduced 5.75-fold (see Additional file 11 for qPCR results).

Nuclear extraction from cells and HAT and HDAC activity assay

The EpiQuik Nuclear Extraction Kit was used according to the manufacturer’s instructions to extract the nuclear proteins from transfected cells.

The Epigenase HDAC activity direct assay kit and the EpiQuik HAT activity kit (Cambridge Biosciences) were used to assess HAT and HDAC activity in transfected cells. The kit is an antibody-based assay with a colourmetric readout, assessed by the Multiskan microplate reader at 450 nm.

Western blotting

Frozen liver tissue was defrosted and homogenised in an approximately equal volume of protein extraction buffer (50 mM Tris-HCl, pH 6.8, 86 nM 2-mercaptoethanol, 2 % sodium dodecyl sulfate (SDS) and general protease inhibitors (cocktail, Sigma, category no. P8340), centrifuged at 13,000 rpm for 15 min and the supernatant containing the soluble protein collected. The protein concentration of the supernatant was determined using a bicinchoninic acid assay according to the supplier's protocol (Abcam, Sigma) adapted to a 96-well plate format. Absorbance at 562 nm was measured and protein concentrations were back-calculated from the standard curve using the SpectroMax software (Molecular Dynamics). Samples were diluted to 5 mg protein ml−1.

SDS PAGE was carried out on a large BIO-RAD kit using a 7.5 % or 12 % SDS-polyacrylamide gel and containing 1.5 M Tris-HCl (pH 8.8). Two SDS-PAGE runs were carried out simultaneously in a standard 0.1 % SDS, Tris-Glycine running buffer. The mini gels were electrophoresed overnight at 45 V. Proteins were then transferred at 350 mA (4 hours) in transfer buffer (20 % methanol, Tris-Glycine buffer) onto a nitrocellulose membrane (Amersham Hybond ECL). Membranes were blocked with 5 % dried skimmed milk (Marvel) dissolved in Tris-buffered saline (TBS; pH 8.8). Membranes were then washed twice for 10 min in changes of TBS before being incubated for 1 hour with a 1/5000 dilution of mouse anti-acetylated lysine antibody (Ac-K-103, Cell Signalling Technologies). Membranes were incubated with the secondary horseradish peroxidise-conjugated anti-mouse antibody (Amersham ECL) at a 1/10,000 dilution in blocking solution (50 ml per membrane) for 1–2 hours. Visualisation of the protein bands was by enhanced chemiluminescence (ECL). The gels were stripped and re-probed with beta actin as a loading gel control.

The developed films were analysed using ChemiImager v4.4 software to give an arbitrary quantitative value to allow comparison of band intensity. This was adjusted to account for background, area and absolute protein concentration (by normalising to the loading gel control intensity).

Data availability

All data have been submitted to GEO under the accession GSE57110.

Ethics approval

The animal study from which the tissue was generated for the current publication was conducted under the Home Office Project Licence 40/2964, held by B.J.M. The work was also reviewed by the University of Liverpool Ethical Committee for Animal Welfare.