RNA Immunoprecipitation of MRB3010-PTP or TbRGG2-HTM

Cell growth and mitochondrial prep (all buffers contain RNase inhibitor)
Lysis of mitochondria (all buffers contain RNase inhibitor)
Preclear
Immunoprecipitation
cDNA and Preamplifcation
Calculations for RIP Analysis (adapted from RNA 2:540 (2018) Suppl. Methods)

Fold enrichment compared to the mock RIP was calculated for each gene of interest (GOI) using the ΔΔCt method comparing the tet uninduced sample to that of the mock IP (that is, comparing the Ct value from the qPCR of the cDNA that was made from the RNA that was isolated in the IP of the tet uninduced experimental sample to the Ct value from the qPCR of the cDNA that was made from the RNA that was isolated the IP of the mock).

Fold change to mock IP = 2(ΔΔCt), where ΔΔCt = (CtGOI-Mock - Ct18S-Mock) - (CtGOI-Uninduced - Ct18S-Uninduced).

To determine the fold change in the RNA associated when MRB7260 was depleted, the RNA detected in the tet-induced sample was compared to the uninduced IP using the ΔΔCt method.

Fold change with MRB7260 knockdown = 2(ΔΔCt), where ΔΔCt = (CtGOI-Uninduced - Ct18S-Uninduced)-(CtGOI-Induced - Ct18S-Induced).

Download Protocol: Natalie RIP Protocol.docx, ΔΔCt spread sheet
(note requires Microsoft Word to open)