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MiRNA Tools. Antimers miRNA Plasmids miRNA duplexes (miRNA mimics) miRNA Viruses miRNA Array Data miRNA qPCR.

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Presentation on theme: "MiRNA Tools. Antimers miRNA Plasmids miRNA duplexes (miRNA mimics) miRNA Viruses miRNA Array Data miRNA qPCR."— Presentation transcript:

1 miRNA Tools

2 Antimers miRNA Plasmids miRNA duplexes (miRNA mimics) miRNA Viruses miRNA Array Data miRNA qPCR

3 Antimers Inhibition in cell culture (293 cells)

4 has-miR-017-5p has-miR-106a has-miR-143 has-miR-195 has-miR-200b has-miR-302c has-miR-302c* has-miR-018 has-miR-019a has-miR-019b has-miR-020 has-miR-021 has-miR-022 has-miR-029b has-miR-092 has-miR-107 has-miR-130a has-miR-187 has-miR-191 has-miR-200a has-miR-204 has-miR-302a has-miR-302a* has-miR-302b has-miR-302b* has-miR-302d has-miR-367 has-miR ul aliquots at 25 uM each. Total 28 antimiRs. Antimers from Rosetta (located in -80--Junlin)

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6 miRNA Plasmids Trnsfxn for overexpression in HeLa Trnsfxn for overexpression in iPS assay (H1SF, MRC5, iMR90)

7 Successfully Cloned miR-372, miR-518b, miR-520c, miR-520f, and control vectors (empty and GFP) Still to be verified (from Liz) miR-367, miR cluster, miR- 130a, miR-512-5p

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10 D6-D21 w/ hES Media (-FGF+Buty+SAHA) on MEFs D0- Infect D2-D6 Expand Cells D2-D6 Expand Cells D(-2) Plate MRC5 D(-2) Plate MRC5 D21- Fix, Stain, Count D6- Trnsfxn D-1- Trnsfxn iPS Trnsfxn Assay (miRNA plasmids or mimics)

11 50%, 0.5µg, 1.5µl L2K70%, 1µg, 2µl L2K 90%, 0.5µg, 1.5µl L2K90%, 1µg, 2µl L2K Plasmid Trnsfxn of H1SF

12 24 hr (D0)48 hr (D1) D5 pUS2-GFP in H1SF

13 D6-D21 w/ hES Media (-FGF+Buty+SAHA) on MEFs D0- Infect D2-D6 Expand Cells D2-D6 Expand Cells D(-2) Plate MRC5 D(-2) Plate MRC5 D21- Fix, Stain, Count D6- Trnsfxn D-1- Trnsfxn iPS Trnsfxn Assay (miRNA plasmids or mimics) No colonies for any assay (H1SF, MRC5, iMR90) Trsnfxn on cells was harsh; cells didn’t look good prior to re-plating into hESC conditions

14 miRNA Oligos (miRNA mimics) Assess small RNA duplex transfxn efficiency by siRNAs (siDcr vs siLuc) Trnsfxn for overexpression in iPS assay (H1SF, MRC5, iMR90)

15 dsCon miR-106a miR-195 miR-17 miR-302c miR-372 miR-373 siLuc siDcr miRNA strands ordered individually by Junlin and Jenn-Yah. Original stocks in -80 (Junlin). Annealed aliquots prepared and diluted to 20µM stocks (Brad -20). siRNA strands ordered individually by Brad. Original stocks in -80 (Brad). Annealed aliquots prepared and diluted to 20µM stocks (Brad - 80 & -20).

16 siRNA Trnsfxn of H1SF

17 D6-D21 w/ hES Media (-FGF+Buty+SAHA) on MEFs D0- Infect D2-D6 Expand Cells D2-D6 Expand Cells D(-2) Plate MRC5 D(-2) Plate MRC5 D21- Fix, Stain, Count D6- Trnsfxn D-1- Trnsfxn iPS Trnsfxn Assay (miRNA plasmids or mimics) No colonies for any assay (H1SF, MRC5, iMR90) Trsnfxn on cells was harsh; cells didn’t look good prior to re-plating into hESC conditions

18 miRNA Viruses Assess miRNA OE following infxn (HeLa) Infxn for overexpression in iPS assay (H1SF, MRC5, iMR90) No colonies

19 miR-372 Virus- constructed by Amy and Wennie. Stocks in -80 (Brad and Wennie) miR-302a-d OE Virus and Control Virus- constructed and purchased from a company by Merav. Stocks in -80 (Wennie)

20 372OE Lentivirus in HeLa (48 hr)

21 miR-302 Cluster Virus in HeLa (48 hr)

22 miRNA Array Data All files from arrays transferred to Kavitha’s computer All array data uploaded to GEO omnibus website

23 miRNA qPCR All kits from Ambion are sorted in -20. Sorting as follows: control RNA (RNUs 66 and 48), mouse miRNAs, hESC miRNAs (302s, ), C19MC miRNA, and other random miRNAs

24 HFF1 50% Seeding 1µg DNA, 2λ L2K 75% Seeding 1µg DNA, 3.5λ L2K 90% Seeding 1µg DNA, 3.5λ L2K

25 HMF2 50% Seeding 1µg DNA, 3.5λ L2K 75% Seeding 3µg DNA, 5λ L2K 90% Seeding 3µg DNA, 5λ L2K


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