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?

Sooner than ...

• Using the lower level of cell number, try to opimize TE and also harvest RNA to make sure enough RNA can be harvested to continue with RT-RT-qPCR

This will have to wait until after the conference since it takes two days to grow up the cells.

• Re-do the activity gel.

It should have worked. Maybe use FRESH 3T3 and Hos samples. Also I am using M1-RNase H1-GFP & M27I-RNase H1-GFP cells.

Brad 11:28, 16 June 2006 (EDT) I am exposing to film right now.

... Later

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

First I need to settle on a TE that I am happy with. So this step is indefinently put off.

• Maybe since the shRNA construct worked so well I should clone it into a miRNA vector and test that?

Perhaps I should retest the shRNA construct once I have optimized the TE. It might do better than 60% endogenous.

Immunolabeling RNase H1, Top3a and POLG

• RNase H1 antibody

I have settled on an antibody and condition for 3T3/Hos cell IF. The antibody can be dilute to 1:150 -> 1:200

• Top3a Antibody

Needs to be extensively tested.

• POLG

Commercial Antibody works great!

What Next?

• Once I get the miRNA construct working ...

I would like to try to modulate RNase H1, and observe the changes in Top3a/POLG/RNase H1 localization. I could use the shRNA construct.

• I have streaked out the bacteria carrying M27I-RNase H1-GFP 3 times, purifed the plasmid, and sent off for sequencing (11:37, 16 June 2006 (EDT)). Hopefully it will come back as M27I, then I will purify close to 200 ug of plasmid and re-transfect. Now that I believe it to be "pure" M27I-RNase H1-GFP, I hope to see only mitochondrial localization!

• I have a plate streaked with various RNase H1-GFP clones (M1, M27I, M27, natural v. optimized nucleotide sequence around the M). I should slowly purify the plasmids and test each one. I will do individually so to be sure not to contaminate one with another.

• When we overexpress RNase H1 in tissue culture

Do we see signs of apoptosis or DNA cleavage? Is it really localizing to the nuclues?