{"text_format":"3","workbook":"xls","n_sheets":1,"sheets":["Sheet1"],"not_rendered":{"external_links":1,"defined_names":1},"read":"whole","date_system":"1904","formulas":"cached_only"}

{"sheet":"Sheet1","index":0,"n_rows":149,"n_cols":3,"rows_kept":149,"flags":["formulas_cached_only"]}
["Sample/Template","details"]
["Source","Cultured human chondrocytes, isolated enzymatically from osteoarthritis patients"]
["Method of preservation","No preservation; Total RNA was extracted directly from PBS washed cell layers"]
["Storage time (if appropriate)","RNA was stored at -80°C and reverse transcribed into cDNA within one month. cDNA was stored at -80°C and analyzed within 6 months."]
["Handling","----"]
["Extraction method","Column based extraction method (NucleoSpin RNA II Kit (Macherey-Nagel, D))."]
[" RNA: DNA-free","All primer pairs used are intron-spanning primers. The RNA extraction protocol included on-column DNA digestion. Using microfluidics (Bioanalyzer), the quality of each RNA preparation was verified. 'Minus RT controls' were prepared from random RNA samples."]
["Concentration","All RNA preparations were quantified using microfluidics (Bioanalyzer)."]
["RNA: integrity","Integrity of RNA each preparation was verified using microfluidics (Bioanalyzer)."]
["Inhibition-free","Not all samples were tested for the absence of inhibitors. However, a representative set of samples was used to control for the absence of inhibitors using dilution series of target genes."]
["Assay optimisation/validation"]
["Accession numbers"]
["MGAT1","NM_002406"]
["MGAT2","NM_002408"]
["MGAT3","NM_002409"]
["MGAT4A","NM_012214"]
["MGAT4B","NM_014275"]
["MGAT5","NM_002410"]
["MGAT5B","NM_144677"]
["MAN1","NM_020379"]
["MAN2","NM_002372"]
["FUT8","NM_178155"]
["GALNT","NM_020474"]
["B3GNT","NM_006577"]
["GCNT1","NM_001490"]
["FUT1","NM_000148"]
["3OST","NM_005114"]
["ST6GAL1","NM_003032"]
["ST6Gal2","NM_032528"]
["ST3Gal3","NM_174963"]
["ST3Gal4","NM_006278"]
["ST3Gal6","NM_006100"]
["ST3GAL1","NM_003033"]
["ST3GAL2","NM_006927"]
["ST6GalNAc1","NM_018414"]
["ST6GalNAc2","NM_006456"]
["ST6GalNAc3","NM_152996"]
["ST6GalNAc4","NM_175039"]
["B4GALNT3","NM_173593"]
["GAPDH","NM_002046"]
[]
["Amplicon details"]
["MGAT1","142 bp"]
["MGAT2","147 bp"]
["MGAT3","132 bp"]
["MGAT4A","176 bp"]
["MGAT4B","120 bp"]
["MGAT5","197 bp"]
["MGAT5B","79 bp"]
["MAN1","204 bp"]
["MAN2","104 bp"]
["FUT8","98 bp"]
["GALNT","177 bp"]
["B3GNT","175 bp"]
["GCNT1","127 bp"]
["FUT1","99 bp"]
["3OST","104 bp"]
["ST6GAL1","116 bp"]
["ST6Gal2","95 bp"]
["ST3Gal3","113 bp"]
["ST3Gal4","88 bp"]
["ST3Gal6","102 bp"]
["ST3GAL1","93 bp"]
["ST3GAL2","115 bp"]
["ST6GalNAc1","95 bp"]
["ST6GalNAc2","125 bp"]
["ST6GalNAc3","97 bp"]
["ST6GalNAc4","96 bp"]
["B4GALNT3","144bp"]
["GAPDH","165 bp"]
[]
[]
["Primer sequence"]
[null,"forward","reverse"]
["MGAT1","GGTGGAGTTGGTGGGTCATC","GAGGAGAGGTCTTGCTTGCC"]
["MGAT2","CTGCTGCTGGACTCACTTC","AATGCTGAAAGGAAAGAACACC"]
["MGAT3","ATGGAGGAGAAGAGAATGGATATG","GAGGGAAACAGGAGCAAGC"]
["MGAT4A","CTTCCTTCCTTACTTTGTCTTGG","TGCTCCTACACGCTTGAAC"]
["MGAT4B","AGACGGAGACGGCAATCG","AGGTGTGGCAGGTGATGG"]
["MGAT5","GAACAAACTCAACCAACTCCAC","CACTCCATAGTCTGCGTAGC"]
["MGAT5B","GGAAGATAATGGTCAGAAGATGC","AGAGTGAAGAAGCCGATGC"]
["MAN1","CCCGCTCAGACACCAAAC","TCCGACAGTATTTCTCCAAGG"]
["MAN2","GACTCTTACTCTCCTGATACCTTC","TACCTTGGCTCCGCACTC"]
["FUT8","GGTTCAGCGGAGAATAACATATC","GACAGCCATAGCCACAGC"]
["GALNT","TAATTTCTCCAGGTGTTACAAAGG","ACTGATTCGTTTCCACATTTCG"]
["B3GNT","AATAAAGTTGTTGGGTATCCTGATG","CTTCTCTTGCTCTCGGTTCC"]
["GCNT1","CAGAGCAGGTAGCAAGGC","TTGGCACAGGTCACATCTTC"]
["FUT1","AACTCTCCCTCTGGTCTACTC","TGGTGAATGCTTGCTCTGG"]
["3OST","CCTCGGCGATTCCTTGTC","TCACTAACTGCTTCTCCATCC"]
["ST6GAL1","AGGTGTGCTGTTGTGTCG","TGTTGGAAGTTGGCTGTGG"]
["ST6Gal2","CGTGGTTATGAGAAAGATGTTGG","GAACTGTCAATGAAGTGATGGC"]
["ST3Gal3","GAATCCTCAACCCATATTTCATCC","CACTGCCACACTGCCAAG"]
["ST3Gal4","TTGGCTCTGGTCCTGGTC","ACGGCTCCTTCTTCTCTGG"]
["ST3Gal6","ATTCCTGAGTGCTGTCTTCC","ATCTTATTTCTCCGTTTCATTTCC"]
["ST3GAL1","CATCCTCTCGGTCATCTTCTC","CAGTAGTGGTGCCAGTTCC"]
["ST3GAL2","GGACGGGCACAACTTCATC","CAGGTTCTTGGCACTCTCAG"]
["ST6GalNAc1","TCTGGCTGTCCTGGTCTTC","TAATGTTCTCTGTGCGTTGATG"]
["ST6GalNAc2","CCAAGTCATCGCCTCCAC","TCAGAATGCCTCCGTTGC"]
["ST6GalNAc3","GATACCATTCTCCTACACATACAG","AAGGTCACAGTCCAGTTGC"]
["ST6GalNAc4","GGCTTTGAGGCGGATGTG","CTGGAAGTAGTGTGAATAGTTGC"]
["GAPDH","GGAGTCCACTGGCGTCTTCAC","GAGGCATTGCTGATGATCTTGAGG"]
[]
["Probe sequence*","no probes used"]
["In silico","All primers were subjected to BLAST analysis"]
["empirical","Primers were used at 100 nM"]
["Priming conditions","For cDNA synthesis, random RT primers were used as provided by the manufacturer (High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Austria)). "]
["PCR efficiency","qPCR efficiencies were determined using dilution series of cDNA prepared from chondrocyte mRNA, unless low expression levels did not allow to generate dilution curves. Used efficiencies are given below in %."]
["MGAT1",103.6]
["MGAT2",96.4]
["MGAT3",105.3]
["MGAT4A",100]
["MGAT4B",100]
["MGAT5",100]
["MGAT5B",100]
["MAN1",97]
["MAN2",107.7]
["FUT8",102.4]
["GALNT",102.3]
["B3GNT",110]
["GCNT1",94.9]
["FUT1",99.1]
["3OST",92.6]
["ST6GAL1",103.6]
["ST6Gal2",100]
["ST3Gal3",98.5]
["ST3Gal4",100]
["ST3Gal6",100]
["ST3GAL1",98.5]
["ST3GAL2",100]
["ST6GalNAc1",100]
["ST6GalNAc2",100]
["ST6GalNAc3",100]
["ST6GalNAc4",106]
["GAPDH",97]
[]
["Linear dynamic range","The dynamic range of the primers was spanning unknown targets as determined by dilution curves."]
["Limits of detection","LODs were not determined in this study."]
["Intra-assay variation","The intra-assay variation was below one Cq."]
["RT/PCR"]
["Protocols"," RT: equal RNA quantities were reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Austria; LOT#: 4368814).                                                                      qPCR: reactions were performed in 25 µl reaction mixtures containing 1 µl cDNA, 12.5 µl SensiMix SYBR Green Master Mix (GenXpress, Austria; LOT#: SMP-110E), 100 nM primers (Metabion, Martinsried, D), and nuclease-free water to 25 µl, and run in duplicate on a StepOnePlus QPCR system (Applied Biosystems)."]
["Reagents","see above. "]
["Duplicate RT","Replicates of the RT step were not performed in this study."]
["NTC","NTCs were included in each run. The absence of both amplification and melting curve peaks were mandatory for the acceptance of the run."]
["NAC","Minus RT controls were prepared from randomly selected RNA samples."]
["Positive control","Inter-run calibrators were not used."]
["Data analysis"]
["Specialist software","Data were analyzed using the StepOnePlus software (Applied Biosystems) considering both amplification efficiencies and normalization to GAPDH as reference gene. "]
["Statistical justification","Primary chondrocytes (PCs) were isolated from 5 donors and cultured and analyzed seperately. All qPCR reactions were performed in duplicate. Data were exported to Microsoft Excel and analyzed using the integrated statistic tools. p-values < 0.05 were considered significant."]
["Transparent, validated normalisation","mRNA expression levels were calculated as relative copy numbers with respect to the expression of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) set at 1000. "]