RNA Detection

Add 375μL RNA ISO buffer and 375μL PCA. Mix gently by several inversions. Spin at max speed (16,100g)at4C for 10 min. Transfe ...

Heat RNA solution to 65C in the thermomixer for 2 min to disrupt secondary structures. Place samples on ice until use instep 9 ...

3.3.1 Bead Washing 1. For each reaction, pipet 225μL of Ribo-Zero magnetic beads into a 1.75 mL centrifuge tube. Pipet slowly to ...

If there are residual beads in the supernatant, repeat the mag- netic separation. Place the RNA on ice and immediately proceed ...

Transfer reactions to 1.75 mL microcentrifuge tubes. Add 100μL of phenol–chloroform–isoamyl alcohol (25:24:1) pH 6.7 to each re ...

Cool the reactions and the RT master mix to 50C for 5 min. Important: Perform steps 5 and 6 while the RNA/primer mix and the R ...

Add 100μL of the mixed beads to each 100μL of sample (see Note 17). Mix thoroughly by pipetting>10 times. Vortex gently. Inc ...

Aliquot 20μL of the PCR reaction mix into eight separate 0.2 mL nuclease-free PCR tubes. Amplify via the following protocol (se ...

Incubate the tubes at room temperature for 2 min. Place the tubes on the magnetic stand for 5 min. Transfer the clear supernata ...

4 Notes The SDS in the buffer may precipitate at room temperature. If it does, heat the buffer very briefly in a 37C water buf ...

Use a 1:1 bead-to-reaction ratio (by volume) to efficiently remove the second strand synthesis oligo. We most often run this PC ...

Lapointe CP, Wilinski D, Saunders HA, Wickens M (2015) Protein-RNA networks revealed through covalent RNA marks. Nat Methods 12 ...

Chapter 31 RAP-MS: A Method to Identify Proteins that Interact Directly with a Specific RNA Molecule in Cells Colleen A. McHugh ...

In recent years, methods such as cross-linking and immunopre- cipitation (CLIP) have been developed that have become the gold st ...

cross-link zero-distance interacting RNA and protein partners, followed by capture of the RNA of interest through hybridization ...

2 Materials Prepare all solutions using ultrapure water and the highest possible purity reagents. Whenever possible, use certifi ...

q-PCR and/or Agilent Bioanalyzer. TurboDNase with high salt tolerance. Glass dounce homogenizer, 2 mL size. UV cross-linker wit ...

Sequencing grade modified trypsin (e.g., Promega). Lysyl endopeptidase, mass spectrometry grade (e.g., Wako). Liquid nitrogen. ...

1Hybridization buffer: 10 mM Tris–HCl pH 7.5, 5 mM EDTA, 500 mM LiCl, 0.5% DDM, 0.2% SDS, 0.1% sodium deoxycholate, 4 M urea, ...

Remove supernatant and resuspend cells in cold PBS to a final concentration of 50 million cells per 1 mL of buffer, pipetting g ...