When isolating Total RNA from cell or tissues, there is a mixture of RNA species. This includes a majority of rRNA for processing of transcripts. You are interested in the mRNA fraction, I assume, therefore you want to decrease the interference of competing species depending on what system you are using RNAseq, etc. This will allow you to calculate concentration more accurately for the each read when preparing your library.
There are kits available that have rRNA depletion protocols you can follow. Hope this helps as a general overview from my experience.
Tim
My strong suggestion is to ask these questions and to work closely with people who will run sequencing for you (I assume, you are not going to load your samples on sequencing machine yourself....). Talk to people directly! They (these people) should help you to design your experiment, choose reagents properly, etc.
I don't think anybody here have an ability to help you better and faster.
Hi Aqleem,
Your first question, regarding how to generate libraries for sequencing:
First you have to decide on what sequencing platform (instrument) you will be sequencing, as this will influence which kit you will use. There are several different commercially available RNA-seq library prep kits on the market. Some are more streamlined than others and some are more efficient in generating libraries from various inputs. Establish how much material you have to work with. If you have limited material, start you search for a low input kit.
As for your second question, regarding rRNA depletion:
As Timothy states, total RNA is made up of several different species of RNA. The greatest part of this will be rRNA. To get rid of the rRNA you can either digest it enzymatically or you can capture the rRNA. Both methodes have pros and cons. I prefer the enzymatic method as this is mostly independent of the quality of the total RNA. Degraded rRNA might slip through if you do a capture method. By performing rRNA depletion you will only get rid of rRNA and you will be left with all other RNAs, including mRNA, small RNA etc.
If you are only interested in mRNA, I would suggest mRNA capture, as this ensures that you not only get rid of rRNA but all other RNAs in the mix.
Again there are several kits on the market. The first decision will be to think of if you want to do mRNA capture or rRNA depletion.
Then, as Olga says, it might be the best to think about what you want from the data before you dive headfirst into ordering kits and reagents.
Happy sequencing,
Chris
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