Supplementary File S1 Genes and primers used in the GeXPhSIRTNADPlex multiplex assay
Supplementary File S2 - Design and optimisation of the hSIRTNADplex
Supplementary File S3 – Inter-individual responses to SIT and interactions with the sirtuin/NAD system
Supplementary File S1 Genes and primers used in the GeXPhSIRTNADPlex multiplex assay
Gene Symbol Gene ref sequence Left primer sequence Right primer sequence Product size Reverse primer
with universals concentration
(nM)
HIC1 / CCDS42230 / CGTGCGACAAGAGCTACAAG / AACTTCTTCCCGCAGATGG / 137 / 500.00SIRT7 / CCDS11792 / TGTCTCAGAACTGTGACGGG / GGAACGCAGGAGGTACAGAC / 143 / 500.00
SIRT4 / CCDS9194 / AGGTCAGAAAAAGTGGGGCT / CTACGAAGTTTCTCGCCCAG / 149 / 500.00
CD38 / CCDS3417 / GCCAAAGTGTATGGGATGCT / TTGTTGCAAGGTACGGTCTG / 155 / 15.60
NMRK2 / CCDS12115 / CGCAACTACACAGTCCCTGA / ACTTCATGCCGTCCAGGTAG / 161 / 500.00
UBE2D2 / NM_181838 / CAGCACAGTGTTCAGCAGGT / TGAAGGGGTAATCTGTTGGG / 172 / 1.90
NMRK1 / CCDS6650 / ACCTCCCAAATTGCAGTGTC / AGCAGGAAATGGCTGACATC / 178 / 500.00
NMNAT3 / CCDS3111 / AGAAGTTTGGCTTGGTGTGC / GCCTGATGTATGTGGCACTG / 184 / 500.00
NMNAT2 / CCDS1354 / GTGGAGCGTTTCACCTTTGT / CACCTCCATATCTGCCTCGT / 190 / 1500.00
PNP / CCDS9552 / GTGAGCTACAGGAAGGCACC / AGCCAAAGACTCGAAGTCCA / 196 / 31.25
SIRT5 / CCDS4527 / CAGGAAAAGGTGCTCCAGAA / CGAGCTCTCTGTCAACCTCC / 202 / 500.00
PSMB6 / D29102 / GAACAACCACTGGGTCCTAC / ACCAGTGGAGGCTCATTCAG / 209 / 500.00
NADSYN1 / CCDS8201 / GGAAGCCACCACATCAGTCT / GAGCAAACAGATAGGCGAGG / 215 / 31.25
NMNAT1 / CCDS108 / GGGTCATCATGGCAGAACTT / CTCTTCCTTCCAGGCCTTTC / 221 / 500.00
SIRT6 / CCDS12122 / CCAAGTTCGACACCACCTTT / TTGGCACATTCTTCCACAAA / 227 / 500.00
PPIB / NM_000942 / CGTCTTCTTCCTGCTGCTG / GCCAAATCCTTTCTCTCCTG / 236 / 15.60
SIRT1 / NM_012238 / GGATTTGGGACTGATGGAGA / CACCTTTCTGGTTTCCTTGC / 245 / 31.25
TDO2 / CCDS34086 / GGCAGCGAAGAAGACAAATC / CAGAATCCAACTCCCAGAGG / 251 / 1500.00
SIRT2 / CCDS12523 / TCGCAGAGTCATCTGTTTGG / GTGACAGATGGTTGGCTTGA / 257 / 500
QPRT / CCDS10651 / ACTTCAAGCCAGAGGAGCTG / TAGTGGATTTTGGGCACTGG / 263 / 1500.00
NAPRT1 / CCDS6403 / TCGTCACTTCCTTTTCAGGC / ACGCTGTAGGTGTCCAGGAG / 269 / 500.00
ABCA1 / CCDS6762 / CATCAAGGGCATCGTGTATG / GAAGCACTGCAGGATTGTCA / 275 / 500.00
SIRT3 / CCDS7691 / AGAACATCGATGGGCTTGAG / AAATCAACCACATGCAGCAA / 281 / 500.00
NAMPT / CCDS5737 / GGCCTTGGGATTAACGTCTT / GATGTGCTGCTTCCAGTTCA / 287 / 7.80
PARP1 / CCDS1554 / ACAGGACCGTATATTCCCCC / TTTCCACCTCCTTTTTGGTG / 294 / 500.00
Kan(r) / ATCATCAGCATTGCATTCGATTCCTGTTTG / ATTCCGACTCGTCCAACATC / 325
Reference gene (bold) used for normalisation (assessed using geNorm ( to establish the most stable expressed transcript for normalisation purposes) and calculation of relative gene expression levels. A synthetic internal reverse transcription and PCR amplification control target is also incorporated (italic).
Supplementary File S2 - Design and optimisation of the hSIRTNADplex
The hSIRTNADplex incorporates 22 gene targets, including all 7 human sirtuin genes (SIRT1-7), 15 enzymes involved in conversion of tryptophan, niacin, the novel vitamin, nicotinamide riboside, and metabolic presursors to NAD and metabolism of NAD, together with 3 potential reference genes (PPIB,PSMB6 and UBE2D2) and a synthetic reference messenger RNA transcript (Kan®) for measuring relative quantitation of gene expression and reaction efficiencies respectively. Sequences used for primer assay design using the GenomeLab System were downloaded from the National Center for Biotechnology Information (NCBI) consensus coding sequences (CCDS) project
PPIB,PSMB6 and UBE2D2 were incorporated to be used as potential reference genes for normalisation of target gene expression as these genes have exhibited stable gene expression in previous experiments in our lab (Drew et al., 2014 and unpublished data). The fourth reference gene is an external synthetic reference control transcript Kan® (supplied with the GeXP assay kit, Beckman Coulter, UK) used to spike each reaction.
GeXPhSIRTNADplex primer assay design and optimisation
The GenomeLabeXpress designer GeXP Software (Beckman Coulter, UK) was used to identify suitable gene specific primers for reverse transcription and PCR amplification as previously described (Drew et al., 2011). Reverse PCR primers were designed with a 3’ gene specific sequence and a 5’end consisting of 19 bases of universal priming sequence. The forward PCR primers were designed with a 3’ gene specific sequence and a 5’end consisting of a different 18-nucleotide universal priming sequence. The gene specific primers were designed to generate PCR amplicons that differ in size by 4 – 7 base pairs, ranging in size from 137 – 325 (Supplementary File S1). Primer sequences were evaluated using BLAST searches to ensure specific amplification of the designed PCR fragments. User-defined regions of the listed sequences were selected for primer design where targets were known to be members of a gene family to exclude homologous regions likely to cause mis-priming and aberrant amplification. Primers with universal sequences were purchased from Sigma-Genosys (UK). The hSIRTNADplex was optimised for human blood total RNA (50ng) using procedures described previously (Drew et al., 2011) and the Genome Lab GeXP start Kit (Beckman Coulter) according to the manufacturer’s instructions.
Briefly, individual primer pairs were initially tested using a reverse primer mix (500nM) incorporating the entire set of primers in conjunction with each forward primer (500nM) individually to ensure a single amplicon of the correct size was generated for each of the designed primer pairs using human blood total RNA. Subsequent optimisation of the hSIRTNADplex incorporating multiplexed primer pairs was conducted to characterise primer products obtained in multiplex reactions. Attenuation was then conducted to determine the optimal attenuation factor to generate an appropriate dynamic range of signals for the gene targets in human blood as described previously (Drew et al., 2011). The final primer concentrations for hSIRTNADplex profiling in the tissue types are provided (Supplementary File S1). No template and no reverse transcriptase controls were conducted to ensure the absence of non-specific reaction products.
GeXP PCR reactions products were processed for capillary electrophoresis and fragment separation using a CEQ™ 8800 (Beckman) as described previously (Drew et al., 2011). Following CEQ analysis the raw data was analysed using the Fragment Analysis module of the GenomeLab System software (Beckman). A size exclusion filter appropriate for the custom designed hSIRTNADplex was applied to identify expected size fragments. The fragment data, peak height and peak areas were then imported to the eXpress Analysis module of the eXpress Profiler software (Beckman) and analysed as described previously (Drew et al., 2011). Analysis of the signal intensity data was conducted using geNorm ( to establish the most stable expressed transcript for normalisation purposes.
References
Drew, J.E., Mayer, C-D., Farquharson, A.J., Young,P., et al., Custom design of a GeXP multiplexed assay used to assess expression profiles of inflammatory gene targets in colon normal, polyp and tumour tissue. J MolecDiagn 2011, 13, 233-42.
Drew JE, Farquharson AJ, Horgan GW, Duthie SJ, Duthie GG Stratification of study subjects detected by gene expression profiling reveals differences in postprandial cell defence system responses. MolecNutr Food Res 2014, 58, 2066–79.
Supplementary File S3–Inter-individual responses to SIT and interactions with thesirtuin/NAD system