Question

Design a protocol for isolating mRNA through immunoprecipitation

Answer 1

RNA IMMUNOPRECIPITATION is an antibody-based technique to map in vivo RNA Protein interaction and modifications in RNA.

protocol maintained for RNA IMMUNOPRECIPITATION is as follows-

1. Harvest cells, with the help of formaldehyde, can achieve cross-linking (if required) , now harvest cells by trypsinization and reharvest using phosphate buffer solution+ nuclear isolation buffer + water

[at least one negative control setup should be prepared along with experiment]

[15 to 20 mins are sufficient but must be optimized]

2. pellet nuclei by centrifugation and suspend them in buffer solution

[avoid any kind of contamination with RNA by using RNAse enzymes ]

[for lysis and settling around 30 mins and for DNase treatment around 30 mins are sufficient]

3. now to shear chromatin use a Dounce homogenizer with around 20 strokes and pellet nuclear membrane and debris using centrifugation

4. for immunoprecipitation 1st incubate supernatant with antibody (against the protein of interest) following this added protein A/G beads and incubate for 1 hour at 4-degree celsius.

[amount of antibody added and incubation time must be optimized, time can range from 3 hours to overnight]

5. we have to remove unbound material through repeated washing cycles. for that 1st pellet beads by centrifugation remove supernatant and resuspend beads in RIP buffer. it should be repeated 3 times followed by one wash in phosphate buffer saline.

6 fir elution or purification of RNA 1st resuspend beads in TRIzol RNA extraction reagent and elute RNA with nuclease-free water .eluted RNA can be stored at -80 degree celsius and protein can be analyzed using western blot

(if formaldehyde-based cross-linking has been done now it should be reversed)

[time for elution is around 30 minutes and cross-link reversal around 4 hours]

the final step is to perform reverse transcription study of this RNA and thus formed cDNA is analyzed with qPCR