RIN calculation

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RIN calculation

Postby newbie17425 » Mar 23 2017 3:22 pm

Hello,

I recently submitted a paper and one of the reviewer wants to have the RIN number for my RNA. How can I calculate or measure RIN number? I looked on the web but it says I need a software and equipment to calculate. Can it be done without it, as we don't have the necessary software to do the same.

Also, the reviewer says what primers were used to make the cDNA. I didn't use primers (one step qPCR) but used a cDNA kit to generate my cDNA from the RNA. Can I say that I used a two-step qPCR protocol i.e. I generated my cDNA using a kit rather than primers?
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Re: RIN calculation

Postby r.rosati » Mar 23 2017 4:15 pm

newbie17425 wrote:Hello,
I recently submitted a paper and one of the reviewer wants to have the RIN number for my RNA. How can I calculate or measure RIN number? I looked on the web but it says I need a software and equipment to calculate. Can it be done without it, as we don't have the necessary software to do the same.


The RIN algorithm was developed by Agilent and I'm afraid you need both instrument and software to obtain it.
The MIQE Guidelines list the "RIN/RQI or Cq of 3' and 5' transcripts" as "Essential information". Both RIN and RQI are linked to instruments; the "open" alternative would be to amplify 5' and 3' transcripts from your samples.
The rationale behind this information is to guarantee that the observed differences in Cq do not derive from differences in RNA quality among sample groups. Both RIN and RQI directly attest the integrity of RNA; amplification of 5' and 3' transcripts attest the lack of preferential amplification on one side of the mRNA, which is an empirical proxy information that might be sufficient in lack of the former options, or if the RNA is admittedly not intact (i.e. from FFPE tissues) but you still want to show that this is not affecting your data.

newbie17425 wrote:Also, the reviewer says what primers were used to make the cDNA. I didn't use primers (one step qPCR) but used a cDNA kit to generate my cDNA from the RNA. Can I say that I used a two-step qPCR protocol i.e. I generated my cDNA using a kit rather than primers?


I think what they mean is to know if you used oligo-dT or random examers, which is an important information.
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Re: RIN calculation

Postby newbie17425 » Mar 23 2017 4:18 pm

How do I amplify 5' and 3' transcripts from my samples?

Also, how can I find or say that I used "oligo-dT or random hexamers"? I designed my primers using Primer3Plus and obtained the sequences using NCBI and Rat Genome Database. I think I used random hexamers for my qPCR.

Thank you.
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Re: RIN calculation

Postby r.rosati » Mar 23 2017 4:28 pm

Amplifying 5' and 3' transcripts would imply getting new primers and testing all your samples. You might be better off finding someone that has a Bioanalyzer, TapeStation or Experion and ask them to run the RNA samples you have, if you believe they are of good quality.

About random hexamers or Oligo-dT: they come with cDNA kits, and you probably used either one or the other, or a combination of both in your cDNA synthesis reaction. Otherwise you let the RNA self-anneal to prime the reaction. I'm not aware of a fourth option.
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Re: RIN calculation

Postby newbie17425 » Mar 23 2017 4:44 pm

Ok. I will try to find a bio-analyzer. If not can I say that the equipment is not available?

I found the following information from my cDNA kit and it says random hexamer.
"contains the remaining reaction components: reaction buffer, dNTPs, oligo (dT)18 and random hexamer primers."
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