RNA quality and Library prep

Something I was enlightened for my time being in the industrial sector

RNA QC

RIN requirements may vary for different kind of NGS. It is not official but generally depends on the out-sourced company (respective optimized protocol), sample nature (fresh tissues, FFPE, single-cell, low input) and the type of NGS. For example, RNA-Seq for mRNA would require 7 or above, with 10 to be maximum, while it can go as low as 2 for FFPE sample.

The major interpretation of RIN would be the integrity of the RNA sample. Secondary to it is the gDNA contamination.

This is a sample with RIN of 8.6. 4 things to note a. The fragment between 28S(upper thick band) and 18S (lower band) on the gel to the right b. the corresponding peak to the left c. the peak at the left side of the peak chart and d. the rRNA ratio

Compare to the RNA sample with RIN of 9.6. The band smear between 28S and 18S and the peak at the forefront of the peak chart. This peak is called the 5S and corresponds to the signal strength of the lowest band (right above the drawn band in green) gDNA, if any, would appear as a distinct band above 28Son the gel. rRNA ratio is also a top indicator of intact RNA, with 2 to be realistically excellent.

Another obvious read-out would be the slope or the tangent of the 28S peak (the dark green line drawn at the bottom of the said peak). The steeper the line the lower the RIN.

Prep a library

To prep a library is not difficult but is definitely tricky - before you get the grasp of each protocol. The basic principal is to extract the right amount of RNA, purify in the way it ought to be, convert it into DNA library, and have it sequenced. The sequencer is pricey cause it takes care of everything after you have the right DNA library, so all you have to practice on is everything upstream to this.

Each brand has their own protocol, proprietary reagents and working principle, therefore at the end of the day it is about getting familiar with the workflow and practice of a chosen product. Here in this book we focused on mRNA sequencing analyse and therefore a point or 2 here would be sufficient to get you started without too much digression.

The general principle for mRNA seq is quite established already. Extract RNA in high quantity, fish out the mRNA using the polyA tail, thermal fragmentation, reverse transcription, then followed by amplification where tagging happens. The thing to optimise here is the temperature used in fragmentation, which correlated to the specs of the sequencer, and the RNA purification steps, which to ensure all the alcohols are gone during drying up without compromising the nucleotide. In this sense, a xNA quality measurement is better to be properly in place, for example the TapeStation from Agilent. This investment is going to be vital in the optimization stage and equally important in the QC process when the whole prep work becomes a routine.

(optional reading) I used to contract out my experiment

As far as I know a thing or 2 on how to prep my own library, I did not do that when I was with my last company. I was at that moment working on two projects for a rather established pharmaceutical, therefore relatively abundant in terms of funding. So when you need to consider how you are going to proceed, you need to do a cost-performance calculation, or C/P analysis.

In the school, you are assigned for one single project and you are expected to know everything related to that one particular area as the back of your hand, you do every single step on your own. What you have is time, space, and access to equipment. What you don't have is money and alternatives that go against your PI. In industrial setting, what you have and don't have are quite the opposite, and this is somehow why the industry are reluctant in directly taking in well-trained scholar because the calculation in C/P would be quite off. For example, I am working in a super limited time frame with multiple projects going on. To get things done you need to stick with science and knowledge which has no corners to cut, and therefore you got to pay for the technical part to exchange for the time to do actual science. In the case of RNA-Seq, I contracted out the library prep and the sequencing run, I am essentially paying for the FASTQ and the RNA QC. The time that I brought off the shelf enable me to proceed other projects in parallel, freeing up my minds for generating more ideas for other new projects which may or may not be on my plate eventually, with the luxury to concentrate on the data analysis to derive the next step. So it boils down to the balance between meeting deadlines, the quality of outcome, and the value that could translate into commercial value.

Remember, you have research progress form to fill out when you are in the University, but the form that you have to fill in commercial setting is your KPI.