Supplemental Table 1 Primer sequences for cloning and

Supplemental Table 1. Primer sequences for cloning and real-time RT-PCR of mi. RNA precursors. Primer Sequence mi. RNAs Cloning (Forward) Cloning (Reverse) mi. R-7 -1 TAATACGACTCACTATAGGGTTGGATGTTG CTGTAGAGGCATGGCCTGTGC mi. R-7 -2 TAATACGACTCACTATAGGGCTGGATACAG TGCGATGGCACCATTAG mi. R-218 -1 TAATACGACTCACTATAGGGGTGATAATGTAGAAAGCTGCGTGACGTTCC mi. R-218 -2 TAATACGACTCACTATAGGGGACCAGTCGC TGCAGGAGAGCACGGTGCTTTCCG mi. R-21 TAATACGACTCACTATAGGTGTCGGGTAGC TGTCAGACAGCCCATCGACT let-7 f-1 TAATACGACTCACTATAGGGTCAGAGTGAG TCAGGGAAGGCAATAGATTGTATAGTTATCTCC let-7 f-2 TAATACGACTCACTATAGGGTGTGGGATGA CGTGGGAAAGACAGTAGACTGTATAGTTATC let-7 g TAATACGACTCACTATAGGGAGGCTGAGGT TGGCAAGGCAGTGGCCTGTACAGTT let-7 i TAATACGACTCACTATAGGGCTGAGG TAGCAAGGCAGTAGCTTGCGCAGTTATCTC

Supplemental Table 2. Primer sequences for detection of mature mi. RNAs. Primer Sequence mi. RNAs RT PCR Forward PCR Reverse let-7 a GCTCAGAAGTCACACTGAGCAACTAT GGGCGGTGAGGTAGTAGG GAAGTCACACTGAGCAACTATACAAC let-7 b GCTCAGAAGTCACACTGAGCAACCAC Same to let-7 a CACACTGAGCAACCACACAAC let-7 c GCTCAGAAGTCACACTGAGCAACCAT Same to let-7 a TCACACTGAGCAACCATACAAC let-7 d GCTCAGAAGTCACACTGAGCACTATG GCGGAGAGGTAGT GTCACACTGAGCACTATGCAAC let-7 e GCTCAGAAGTCACACTGAGCACTATA CGGGCGGTGAGGTAGG AGAAGTCACACTGAGCACTATACAAC let-7 f Same to let-7 a CGGGCGGTGAGGTAGTAGA AGAAGTCACACTGAGCAACTATACAAT let-7 g GCTCAGAAGTCACACTGAGCACTGTA CGGGCGGTGAGGTAGTAGT AAGTCACACTGAGCACTGTACAAA let-7 i GCTCAGAAGTCACACTGAGCACAGCA CGGGCGGTGAGGTAGTAGT CACACTGAGCACAAA mi. R-24 CACCGTTCCCCGCCGTCGGTGCTGTTC CCCGCCTGGCTCAGTTC CCGTCGGTGCTGTTCCTG mi. R-92 CACCGTTCCCCGCCGTCGGTGCAGGCC CCCGCCTATTGCACTTGTC GTCGGTGCAGGCCGGG mi. R-10 b GACCGTTCCCCGCCGTCGGTCCACAAA CCGCCGTACCCTGTAGAA CGTCGGTCCACAAATTCG mi. R-221 CACCGTTCCCCGCCGTCGGTGGAAACC CGGGCAGCTACATTGTCTG CGTCGGTGGAAACCAGCA mi. R-222 CACCGTTCCCCGCCGTCGGTGACCCAG CGGGCAGCTACATCTGG CGTCGGTGACCCAGTAGC mi. R-21 CACCGTTCCCCGCCGTCGGTGTCAACA CCCGCCTAGCTTATCAGACTG GCCGTCGGTGTCAACATCA mi. R-486 -5 p CACCGTTCCGCGCCGTCGGTGCTCGGG CGCCGTCCTGTACTGAGCT GTCGGTGCTCGGGGCAG mi. R-451 CACGGAACCCCGCCGACCGTGAACTCA CGCCGAAACCGTTACCAT GCCGACCGTGAACTCAGTAAT mi. R-15 b CACCGTTCCCCGCCGTCGGTGTGTAAA CCGCCGTAGCAGCACATC CCGTCGGTGTGTAAACCATG mi. R-146 b-5 p CACCGTTCCCCGCCGTCGGTGAGCCTA CGGGCGTGAGAACTGAATT CGTCGGTGAGCCTATGGA mi. R-128 CACGGAACCCCGCCGACCGTGAAAGAG GGCGTCACAGTGAACCG CGACCGTGAAAGAGACCG mi. R-181 c CACGGAACCCCGCCGACCGTGACTCAC CCGCCGAACATTCAACCT CGACCGTGACTCACCGAC mi. R-181 a Same to mi. R-181 c CGCCGAACATTCAACGC Same to mi. R-181 c mi. R-181 b CACGGAACCCCGCCGACCGTGACCCAC CCGCCGAACATTGC GACCGTGACCCACCGAC mi. R-425 GACCGTTCCCCGCCGTCGGTCTCAACG GGGCGAATGACACGATCAC CGTCGGTCTCAACGGGAG mi. R-7 CACCGTTCCCCGCCGTCGGTGACAACA CGCCCTGGAAGACTAGTGAT CCGTCGGTGACAACAAAAT mi. R-124 CACCGTTCCGCGCCGTCGGTGGGCATT CATACCTAAGGCACGCGG GTCGGTGGGCATTCACC mi. R-137 CACCGTTCCCCGCCGTCGGTGCTACGC CCGCCGTTATTGCTTAAGAA CGTCGGTGCTACGCGTAT mi. R-139 -5 p CACCGTTCCCCGCCGTCGGTGCTGGAG CCGCCTCTACAGTGCACGT CGTCGGTGCTGGAGACAC mi. R-218 CACCGTTCCCCGCCGTCGGTGACATGG TCGGGCTTGTGCTTGATCT CCGTCGGTGACATGGTTAG

Supplemental Table 3. Comparison of stem-loop and linear RT oligonucleotides. mi. RNA mi. R-21 mi. R-7 mi. R-218 let-7 f let-7 g let-7 i RT Oligonucleotide Sequence Tm (°C) ∆GRT (kcal/mol) ∆GPCR (kcal/mol) Stem-loop CACCGTTCCCCGCCGTCGGTGTCAACA 86. 2 -2. 37 0. 19 Linear GACCCTTCGCGGCCGTCGGTGTCAACA 85. 6 -0. 16 1. 21 Stem-loop CACCGTTCCCCGCCGTCGGTGACAACA 86. 2 -2. 98 -0. 55 Linear GACCCTTCGCGGCCGTCGGTGACAACA 85. 6 -0. 16 1. 21 Stem-loop CACCGTTCCCCGCCGTCGGTGACATGG 87. 1 -2. 98 -0. 55 Linear GACCCTTCGCGGCCGTCGGTGACATGG 86. 4 -0. 16 1. 21 Stem-loop GCTCAGAAGTCACACTGAGCAACTAT 71. 0 -3. 22 -0. 20 Linear CGAGAGTCAGAAGTCACACTGAGCAACTAT 70. 9 -0. 02 1. 40 Stem-loop GCTCAGAAGTCACACTGAGCACTGTA 72. 8 -3. 22 -0. 20 Linear CGAGAGTCAGAAGTCACACTGAGCACTGTA 72. 8 -0. 02 1. 40 Stem-loop GCTCAGAAGTCACACTGAGCACAGCA 77. 2 -3. 22 -0. 20 Linear CGAGAGTCAGAAGTCACACTGAGCACAGCA 77. 2 -0. 50 1. 17 The most stable secondary structure was adopted to calculate ∆G for linear RT oligonucleotides. Sequence differences between stem-loop and linear RT oligonucleotides for each mi. RNA are underlined.

Supplemental Fig. 1 Assay Design Flowchart Extract mature mi. RNA sequence (seq) Design RT oligonucleotide 1. 5’ stem-loop tag seq (stem: 5 -6 nt; loop: ~11 nt) without significant homology to sequences from species of interest (eg. homo sapiens) 2. 3’ mi. RNA-specific seq (6 nt, reverse complementary to 3’ of mi. RNA sequence) 3. Optionally, d. U residue can be incorporated in the loop region. Evaluate RT oligonucleotide (mfold) 1. Formation of stable stem-loop secondary structure at RT condition (∆G -0. 5 kcal/mol) 2. Does not form stable stem-loop secondary structure at PCR condition (∆G -0. 5 kcal/mol) Design hemi-nested real-time PCR primer 1. Forward primer (Tm 58°C) with 5’ tag seq to increase Tm and 3’ mi. RNA specific seq (~12 nt) 2. Reverse primer (Tm 58°C) with partial stem-loop tag seq (8 -10 nt) and mi. RNA specific seq (9 -11 nt, including 6 nt used in RT and 3 -5 nt 3’ protruding seq). Example (hsa-mi. R-21) hsa-mi. R-21: 5’- uagcuuaucagacugauguuga -3’ RT Oligonucleotide: (d. U) mi. R-21 specific seq (RT) 5’- CACCGTTCCCCGCCGTCGGTGTCAACA -3’ Stem -3’ loop Stem 5’∆GRT = -2. 37 kcal/mol ∆GPCR = 0. 19 kcal/mol (d. U) Forward Primer: 5’- CCCGCCTAGCTTATCAGACTG -3’ Tag seq mi. R-21 specific seq Reverse Primer: 5’- GCCGTCGGTGTCAACATCA -3’ Reaction Conditions RT: Na+ = 75 m. M, Mg 2+ = 3 m. M, Temp = 42°C PCR: Na+ = 50 m. M, Mg 2+ = 2. 5 m. M, Temp = 60°C Partial stemloop tag seq (RT) (Hemi-nested PCR) mi. R-21 specific seq Supplemental Fig. 1. Flow Chart and example for designing hemi-nested real-time RT-PCR assay.

Supplemental Fig. 2 A B let-7 d let-7 e Supplemental Fig. 2. Quantification of synthetic let-7 d and let-7 e mi. RNA dilutions with U 251 total RNA spike-in. Standard dilutions (109, 108 and 107 copies) of synthetic let-7 d (A) or let-7 e (B) were spiked with 100 ng of total RNA isolated from U 251 cells. Control mi. RNA dilutions or total RNA spiked-in mi. RNA dilutions were reverse transcribed with let-7 d or let-7 e RT primers. The c. DNA samples (10% v/v) were amplified by real-time PCR. Standard curves were plotted as Ct versus Log (Copies of mi. RNA per RT).

Supplemental Fig. 3 A B mi. R-24 109 103 NTC C D mi. R-92 NTC 109 103 E F mi. R-218 NTC 1010 104 Supplemental Fig. 3. Dynamic range and efficiency of mi. R-24 (A, B), mi. R-92 (C, D) and mi. R-218 (E, F) real-time RTPCR assays. Standard dilutions of synthetic mi. R-24, -92 and -218 were reverse transcribed with mi. RNA-specific RT oligonucleotide. The c. DNA samples (10% v/v) were amplified by real-time PCR along with non-template control (NTC). Amplification plots (A, C, E) and standard curves (B, D, F) of the assay were shown. Standard curves were plotted as Ct versus Log (Copies of mi. RNA per RT).

Supplemental Fig. 4 mi. R-7 M a b mi. R-21 c a b mi. R-218 c a b c let-7 f a b let-7 g c a b let-7 i c a b c U 6 100 bp 50 bp 25 bp Supplemental Fig. 4. Gel electrophoresis of mi. RNA real time RT-PCR products. Real time RT-PCR assays were performed with 106 copies of synthetic mi. RNA (a), 10 ng U 251 total RNA (b) or non-template control (c). U 6 was amplified from 10 pg of U 251 total RNA. The amplified products and the 25 bp marker (M) were resolved by 4% agarose gel. Product sizes: mi. R-7 (37 bp), mi. R-21 (38 bp), mi. R-218 (36 bp), let-7 f (45 bp), let-7 g (42 bp), let -7 i (38 bp) and U 6 (94 bp).

Supplemental Fig. 5 A mi. R-21 RT Oligonucleotide Sequence d. T CACCGTTTTCGGTGTCAACA d. U CACCGUUUUCGGTGTCAACA B C d. T d. U – UDG + Pr – Pr Supplemental Fig. 5. UDG treatment of d. U-incorporated RT oligonucleotide prevented it from serving as PCR primer after RT. A) Sequence of standard (d. T) and d. U-incorporated (d. U) RT oligonucleotides for mi. R-21. Synthetic mi. R-21 (109 copies) were reverse transcribed with of d. T (B) or d. U (C) RT oligonucleotides. The c. DNA samples (10% v/v) were then treated with (red amplification curves) or without UDG (blue amplification curves) and subjected to real-time PCR with both forward and reverse primers (+ Pr) or forward primer alone (- Pr).

Supplemental Fig. 6 A 0 5 15 30 60 180 360 U 0126 (5 µM) - - - + + + GDNF (min) 0 5 15 GDNF (min) Phospho-ERK 1/2 Total ERK 1/2 B Phospho-ERK 1/2 Total ERK 1/2 Supplemental Fig. 6. GDNF-induced signaling activation in U 251 human glioblastoma cells. Control and GDNF stimulated U 251 cells were lysed with 2% SDS. Total protein lysates were quantified using micro. BCA protein assay (Pierce, Rockford, IL, USA). Total protein (10 µg) were separated by SDS-PAGE and probed with antibodies against phospho-ERK 1/2 (Cell Signaling Technologies, Danvers, MA, USA). The blots were striped in Western Blot Stripping Buffer (Pierce) and re-probed with total ERK 1/2 (Cell Signaling Technologies) to verify equal loading. Chemiluminescence was imaged analyzed by Chemi. Doc system (Bio-Rad). A) Time-course of ERK 1/2 MAPK activation by GDNF (100 ng/ml) in U 251 cells. B) GDNF-induced ERK 1/2 MAPK activation was inhibited by pre-treatment of MEK inhibitor U 0126 (5 µM).

Supplemental Fig. 7 B let-7 i Fold Change mi. R-218 Fold Change A Time (min) Supplemental Fig. 7. Quantification of GDNF-regulated mi. RNAs by single-plexed and multiplexed realtime RT-PCR. Total U 251 RNA samples were reverse transcribed by single mi. RNA-specific RT oligonucleotide (black lines) or 24 -plexed RT oligonucleotides (red lines). The c. DNA samples were then quantified for expressions of (A) mi. R-218 and (B) let-7 i. Regulation of these mi. RNAs was expressed as fold changes to non-stimulated control samples.