How do I recover RNA from IP sample?

I am working with RNA binding proteins which I am trying to know what are their target mRNA(s). For this, I am doing RIP-Seq. I started by performing an RNA immunoprecipitation using magnetic dynabeads that target my protein of interest (which is attached to their target mRNAs). After I pulled down my protein using the magnetic beads, TRIzol to get the RNA extracted from the beads-protein complex and the Rnaqueous micro kit (ambion) to purify the RNA, my results showed a very low yield of RNA with a lot of Trizol/phenol/salt contamination as you can see below:

Any recommendation of how I can get my RNA isolated from the beads-protein complex? and how I can get a better and pure yield of RNA?

Thank you.

I need an explanation

I am planning to perform immunoprecipitations using the Pierce anti-HA magnetic beads from Thermo Fisher (Catalog number: 88836) and I was wondering whether anybody has incubated these beads with their protein samples at room temperature for long periods of time?

I have used these beads in a previous lab but we would always perform the incubation at 4 degrees on a rotating wheel for 2-3 hours. However, the protocol on the Thermo website suggests doing this incubation for 30 minutes at room temperature.

I will be using these samples for mass spectrometry analysis and so, I was wondering if 30 minutes at room temperature would be long enough for adequate binding of HA-tagged proteins or would longer incubation times give better results?

Any information would be a big help, thank you!

Hello, I'm trying to investigate what type of RNA I'm pulling down from cell lysate when I co-immunoprecipitate a protein complex with Pierce Protein A Magnetic Beads. My research advisor has instructed me to use TRIzol. Could I simply add TRIzol to my Protein A Magnetic Beads+protein complex sample? Thank you for your time.

I am going to run some ChIP assays (qPCR based, not ChIP-seq) and am just wondering how important it is to include RNAse and Proteinase K treatment steps after elution from the beads and before the overnight reverse cross linking step. I have heard some say it is not needed but many protocols include it. Does the Prot-K help with the reverse cross links or protect the DNA by degrading nucleases? Not sure why RNA removal is important, as surely this will not interfere with the final PCR.

Any ideas?

I did an RNA-IP out of cells and my RNA yield for the inputs was great but the yield was poor for the IP samples. Is there a way to increase the yield of RNA? (other than increasing the starting material; I used a confluent 15 cm tissue culture dish per sample)

Hi there!

I'm wondering about using glycogen in RNA extraction? I'm working with low numbers of cells so how i can use glycogen to increase RNA yield? could we use it through kit of extraction? If yes, then in which step?

i need the process in detail.

your answer will be appreciated

Dear all

I wanted to validate some of the points that I have heard about RNA extraction using Trizol methods. For some of them, I could not find justification online. I wanted to know if they are really "precautions" or just a myth.

1. When resuspending cells in Trizol, they can make clumps. Do not pipette vigorously and cause bubbles in the solution. Also, do not use syringes. Is this precaution really true ?

2. Do not vortex after adding Chloroform as that can lead to genomic DNA contamination. Is this precaution really true ?

3. Do not pipette the water/buffer when resuspending RNA pellet. This can shear the RNA ! Is this precaution really true ?

Looking forwards to your responses.