Synthetic microrna Reference Standards Genomics Research Group ABRF 2015 Don A. Baldwin, Ph.D. support@signalbiology.com
Reference samples for Platform evaluation Protocol development Assay service improvement
Randomize Key Name CLUSTALW 1 hsa-mir-664*...acuggcuagggaaaaugauuggau... 2 hsa-mir-577...uagauaaaauauugguaccug... 3 hsa-mir-576-3p...aagauguggaaaa.auuggaauc... 4 hsa-mir-208a...auaagacgagcaaaaagcuugu... 5 hsa-mir-208b...auaagacgaacaaaagguuugu... 6 hsa-mir-559...uaaag.uaaauaugcaccaaaa... 7 hsa-mir-9*...auaaagcuagaua...accgaaagu... 8 hsa-mir-142-5p...cauaaag.uagaaa.gcacuacu... 9 hsa-mir-335...ucaagagcaauaacgaaaaaugu... 10 hsa-mir-620...auggagauagauauagaaau... 11 hsa-mir-516a-5p...uucucgaggaaagaagcacuuuc... 12 hsa-mir-516b...aucuggagguaagaagcacuuu... 13 hsa-mir-1255a...aggaugagcaaagaaaguagauu... 14 hsa-mir-1255b...cggaugagcaaagaaagugguu... 15 hsa-mir-765...uggaggag.aaggaaggugaug... 16 hsa-mir-483-5p...aagacgggaggaaagaagggag... 17 hsa-mir-1...uggaauguaaagaaguauguau... 18 hsa-mir-206...uggaauguaaggaagugugugg... 19 hsa-mir-452*...cucaucugcaaagaaguaagug... 20 hsa-mir-1285...ucugggcaacaaagugagaccu... 21 hsa-mir-1273...gggcgacaaagcaagacucuuucuu... 22 hsa-mir-711...gggacccagggagagacguaag... 23 hsa-mir-423-5p...ugaggggcagagagcgagacuuu... 24 hsa-mir-1286...ugcaggaccaagaugagcccu... 25 hsa-mir-96*...aaucaugugcagugccaauaug............ 877 hsa-mir-621...ggcuagcaacagcgcuuaccu... 878 hsa-mir-1251...acucuagcugccaaaggcgcu... Design of Standards Random selection across range of composition Four pools of 16 Fifth pool of 829 Distribution of selected family members Many thanks to Life Technologies - AB
Name Sequence Pool hsa-mir-130b...cagugcaaugaugaaagggcau... 1 hsa-mir-130a...cagugcaauguuaaaagggcau... 2 hsa-mir-301b...cagugcaaugauauugucaaagc... 3 hsa-mir-301a...cagugcaauaguauugucaaagc... 4 hsa-mir-376a...aucauagaggaaaauccacgu... 1 hsa-mir-376c...aacauagaggaaauuccacgu... 2 hsa-mir-181d...aacauucauuguugucggugggu... 3 hsa-mir-181b...aacauucauugcugucggugggu... 4 hsa-let-7f...ugagguaguagauuguauaguu... 1 hsa-let-7a...ugagguaguagguuguauaguu... 2 hsa-let-7e...ugagguaggagguuguauaguu... 3 hsa-let-7b...ugagguaguagguugugugguu... 4 hsa-let-7c...ugagguaguagguuguaugguu... 1 hsa-mir-98...ugagguaguaaguuguauuguu... 2 hsa-let-7d...agagguaguagguugcauaguu... 3 hsa-let-7g...ugagguaguaguuuguacaguu... 4 hsa-let-7i...ugagguaguaguuugugcuguu... 1 hsa-mir-520b...aaagugcuuccuuuuagaggg... 2 hsa-mir-520c-3p...aaagugcuuccuuuuagagggu... 3 hsa-mir-520f...aagugcuuccuuuuagaggguu... 4 hsa-mir-15b...uagcagcacaucaugguuuaca... 1 hsa-mir-15a...uagcagcacauaaugguuugug... 2 hsa-mir-23a...aucacauugccagggauuucc... 3 hsa-mir-23b...aucacauugccagggauuacc... 4 Target discrimination Related sequences assigned to separate pools, should be measured at different abundances within a Standard
Preparation of Standards Request protocol from Don support@signalbiology.com Request shipment of pools Send a prepaid shipping label, or your address and FedEx account number Mix pools to create Standards A-E Standard Pool 1 Pool 2 Pool 3 Pool 4 Pool 5 (majority) A B 0.5x 0. 0.0 0.00 C 0. 0.0 0.00 0.5x D 0.0 0.00 0.5x 0. E 0.00 0.5x 0. 0.0 Total micrornas 16 16 16 16 840 = 0.5 nm each mirna Each Standard contains 3 ng/ul
Affymetrix mirna GeneChip Sridar Chittur, SUNY Albany One chip per Standard RMA background correction No inter-chip normalization Calculate pool averages for each Standard Average signal Pool dilution
Affymetrix mirna GeneChip Discrimination of related mirnas Signal Standard Name Sequence Pool hsa-mir-130b...cagugcaaugaugaaagggcau... 1 hsa-mir-130a...cagugcaauguuaaaagggcau... 2 hsa-mir-301b...cagugcaaugauauugucaaagc... 3 hsa-mir-301a...cagugcaauaguauugucaaagc... 4
Affymetrix mirna GeneChip Cross-hybridization of related mirnas Signal Standard Name Sequence Pool hsa-mir-520b...aaagugcuuccuuuuagaggg... 2 hsa-mir-520c-3p...aaagugcuuccuuuuagagggu... 3 hsa-mir-520f...aagugcuuccuuuuagaggguu... 4
HTG Molecular Bruce Seligmann Debrah Thompson HTG EdgeSeq Standards spiked into yeast trna, 75 pg input Nuclease protection assay Quantitation by probe sequencing Bowtie2 alignment to mirbase mature mirnas in probe panel
ABRF reference mirna Latin Square Test 0.5x 0.5x 0.5x 0. 0.0 0.00 0. 0.0 0.00 0.0 0.00 0. 0.5x 0.00 0. 0.0 mirbase 19 assay
Specificity differentiating between closely-related mirnas Pool-specific signals (using all probes) 1-base difference 2-base difference 6862 total counts 25547 27834 total counts 174 4425 810 95 Pool 3 Pool 4 mir-301a-3p 30 mir-301b hsa-mir-301b CAGUGCAAUGAUAUUGUCAAAGC hsa-mir-301a CAGUGCAAUAGUAUUGUCAAAGC Pool 2 Pool 3 let-7e-5p let-7a-5p hsa-let-7a UGAGGUAGUAGGUUGUAUAGUU hsa-let-7e UGAGGUAGGAGGUUGUAUAGUU EdgeSeq differentiates between closely-related mirnas (1 or 2 base differences) in different pools, even within a complex background.
Illumina Small RNA Sequencing Catharine Aquino Hubert Rehrauer Functional Genomics Center Zurich Standards only, no background TruSeq Small RNA Library Prep Kit HiSeq 2500 Alternative library production Adam Morris Bioo Scientific No 5 phosphates on the oligomers
Illumina Small RNA Sequencing
Illumina Small RNA Sequencing Read count Mapped length
Illumina Small RNA Sequencing Read count Mapped length
Illumina Small RNA Sequencing Read count Mapped length
Work to be done Phosphorylate the pools by kinase reactions Repeat TruSeq Small RNA sequencing Quantitative PCR TaqMan Array MicroRNA Card, Nadereh Jafari