In Vivo Processing Assay Based on a Dual-Luciferase Reporter System to Evaluate DROSHA Enzymatic Activity - miRNA Maturation: Methods and Protocols - page 85

1. Dulbecco's modified Eagle's medium (DMEM).
2. Fetal bovine serum (FBS).
3. 1× Phosphate-buffered saline (PBS, pH 7.4).
4. 5× Trypsine. Dilute to 1× solution in PBS and store at 4 °C.
5. HEK293T cells maintained in DMEM supplemented with
10 % FBS at 37 °C with 10 % CO 2 in 75 cm 2 flasks.

1. TransIT ® -LT1 Transfection Reagent (Mirus Bio LLC, USA).
Store at −20 °C.

2.2 Transfection
Reagents

1. pMir-Report Luciferase (Ambion, Austin, USA).
2. pSuper (Oligoengine).
3. pRL-CMV (Promega Inc., USA).
4. pMir-Report Luciferase pri-miR-16-1wt (amplified from
genomic DNA with primers (for sequences see Subheading 2.5 ,
Table 2 ) and cloned with Hind III into pMir-Report Luciferase).
5. pMir-Report Luciferase pri-miR-16-1ss (“ss” meaning “single
stranded”; mutated using primers (for sequences see
Subheading 2.5 , Table 2 ) and cloned with Hind III into pMir-
Report Luciferase).
6. pSuper_shEGFP (synthetic oligos ( see Subheading 2.4 ,
Table 1 ) were cloned between the BglII and HindIII sites of
pSuper plasmid, Oligoengine).
7. pSuper_shDROSHA (synthetic oligos ( see Subheading 2.4 ,
Table 1 ) were cloned between the BglII and HindIII sites of
pSuper plasmid, Oligoengine).

2.3 Plasmids

2.4 Oligos for shRNA
Plasmids

Table 1
shRNA sequences used for cloning into pSuper plasmids

Name Sequence (oligoengine)

shDROSHA_s 5′-GATCCCCGCGAGTAGGCTTCGTGACTT

ATATGACACCTGACCCATGTCATATAA
GTCACGAAGCCTACTCGTTTTTA-3′

shDROSHA_as 5′-AGCTTAAAAACGAGTAGGCTTCGTGAC

TTATATGACATGGGTCAGGTGTCATAT
AAGTCACGAAGCCTACTCGCGGG-3′

shEGFP_s 5′-GATCCCCGCTGACCCTGAAGTTCATCT

TCAAGAGAGATGAACTTCAGGGTCAGC
TTTTTA-3′

shEGFP_as 5′-AGCTTAAAAAGCTGACCCTGAAGTTCAT

CTCTCTTGAAGATGAACTTCAGGGTCA
GCGGG-3′