Quantifying miRNA and mRNA Levels

Quantitive-RT-PCR for evaluating miR-221, miR-222 and PUMA mRNA levels was performed as previously described (12 (link)). The primers for miR-221(#000524), miR-222(#000525), U44(#001094), PUMA(#Hs00248075_m1), and GAPDH (#Hs99999905_m1) were purchased from Applied Biosystems (ABI). Immunoblots were obtained as previously described (13 (link)). Primary antibodies included: anti-PUMA (#4976, Cell Signaling), anti-BAX (#2772, Cell Signaling), anti-Cleaved Caspase 3 (#9661, Cell Signaling), anti-PARP (#9542, Cell Signaling), anti-BID (#sc-11423, Santa Cruz), anti-BAK (#06-536, Millipore), anti-P53 (#SC-126, Santa Cruz) and anti-BIM (#202000, Calbiochem). Horseradish peroxidase (HRP)-conjugated secondary antibodied included: anti-rabbit IgG (#W4011, Promega), anti-mouse IgG (#W4021, Promega), and anti-Actin (#sc-1615, Santa Cruz).

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Zhao J.J., Chu Z.B., Hu Y., Lin J., Wang Z., Jiang M., Chen M., Wang X., Kang Y., Zhou Y., Ni Chonghaile T., Johncilla M.E., Tai Y.T., Cheng J.Q., Letai A., Munshi N.C., Anderson K.C, & Carrasco R.D. (2015). Targeting the miR-221-222/PUMA/BAK/BAX pathway abrogates dexamethasone resistance in multiple myeloma. Cancer research, 75(20), 4384-4397.

Publication 2015

GAPDH anti-PUMA anti-BAX anti-Cleaved Caspase 3 anti-PARP anti-BID anti-BAK anti-P53 anti-Actin

Actin Anti igg Antibodies Caspase 3 Gapdh Horseradish peroxidase Immunoblots Mouse Mrna Primers Promega Puma Rabbit Rt pcr

Corresponding Organization : Brigham and Women's Hospital

Other organizations : Union Hospital, Huazhong University of Science and Technology, Tongji University, Moffitt Cancer Center, VA Boston Healthcare System

Top 5 similar protocols

Variable analysis

independent variables

MiR-221
MiR-222
PUMA mRNA

dependent variables

MiR-221 levels
MiR-222 levels
PUMA mRNA levels
PUMA protein levels
BAX protein levels
Cleaved Caspase 3 protein levels
PARP protein levels
BID protein levels
BAK protein levels
P53 protein levels
BIM protein levels

control variables

GAPDH

positive controls

None specified

negative controls

None specified

Annotations

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