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RISC effect to mRNA during RNA-seq preparation
Hello,
I have a question regarding the RISC (RNA induced silencing complex, especially miRNA) and RNA-seq. I'm not a biologist so forgive me if I have wrong interpretation. Let's start with what I have in mind.
So, what I think about RISC is that RISC will bind to mRNA. To simplify, I will write mRNA+RISC. What I want to understand is, what will happen with mRNA+RISC during RNA-seq preparation.
To understand this, I need to understand how RNA-seq, especially Illumina TruSeq v2 protocol works but I'm not biologis so I don't know biochemistry that much so my understanding is limited. This is what I understand what will happen with mRNA-RISC.
First possibility is mRNA+RSIC will not be sequenced. I think this because with the RISC bound to mRNA, mRNA will not be able to react to chemical reaction.
Second possibility is mRNA+RISC still react to the tagging, and any other step until the fragmentation step. At the amplification step, mRNA+RISC will be fragmented. There will be some fragment which has no RISC bound to it so these kind of fragment can be amplified and the cDNA result can be sequenced. The fragment which has RISC bound to it will not be amplified because the RISC will prevent the cDNA binding to amplify the fragment. The consequences of this result is that there will be a low reads for area which RISC bound to mRNA.
To illustrate this, suppose a mRNA length is 1000 nucleotide. RISC bound to 300-320 bp. What I expected is the aligned reads around 300-320 will be relatively low in coverage compare to other parts of the mRNA. What do you think about this?
I have a question regarding the RISC (RNA induced silencing complex, especially miRNA) and RNA-seq. I'm not a biologist so forgive me if I have wrong interpretation. Let's start with what I have in mind.
So, what I think about RISC is that RISC will bind to mRNA. To simplify, I will write mRNA+RISC. What I want to understand is, what will happen with mRNA+RISC during RNA-seq preparation.
To understand this, I need to understand how RNA-seq, especially Illumina TruSeq v2 protocol works but I'm not biologis so I don't know biochemistry that much so my understanding is limited. This is what I understand what will happen with mRNA-RISC.
First possibility is mRNA+RSIC will not be sequenced. I think this because with the RISC bound to mRNA, mRNA will not be able to react to chemical reaction.
Second possibility is mRNA+RISC still react to the tagging, and any other step until the fragmentation step. At the amplification step, mRNA+RISC will be fragmented. There will be some fragment which has no RISC bound to it so these kind of fragment can be amplified and the cDNA result can be sequenced. The fragment which has RISC bound to it will not be amplified because the RISC will prevent the cDNA binding to amplify the fragment. The consequences of this result is that there will be a low reads for area which RISC bound to mRNA.
To illustrate this, suppose a mRNA length is 1000 nucleotide. RISC bound to 300-320 bp. What I expected is the aligned reads around 300-320 will be relatively low in coverage compare to other parts of the mRNA. What do you think about this?
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