Holmesbr/plan

Introduction

This page is meant to keep my two mentors up-to-date on what I am working on and also help keep myself organized.

Anyone can post if they have helpful suggestions.

Specific Aims

RNAi Knockdown of RNase H1 in situ

What I have done

• miRNA

I have 4 miRNA constructs plus a negative control miRNA construct. I have tested these against endogenous RNase H1 in 3T3 cells, but the knock-down by RT-RT-qPCR has been abysmal, with only one construct acheiving ~40% knock-down. However, this may be due to poor Transfection Efficiency.

• Transfection Efficiency

The latest results (6/14/6) indicate that maybe I am plating 4X too many cells on Day 0 before transfecting (by FACS analysis, I was able to increase the TE from 50%to 80%).

• Analysis of RNase H1 Knock-down by RNase H1 Activity Gel (aka Renaturation Assay)

I harvested a known quanity of Hos and 3T3 cells 5 days previous, pelleted and froze at -20. I ran a seriel dilution of these sample as well as 7.6 and .76 ng of RNase H1 as a positive control. I incubated one night, and showed 0 RNase H1 activity, even in the positive control. I incubated a second night. Still no RNase H1 activity.

What Next?

• Test miRNA on Exogenous RNase H1 by co-transfecting with M27-RNase H1-GFP

• Using the lower level of cell number, try to opimize TE and also harvest RNA to make sure enough RNA can be harvested.

• Re-do the activity gel. It should have worked. Maybe use FRESH 3T3 and Hos samples.