Hello Fellow researchers !!!!
I have been trying to isolate mRNA from extracted RNA (Alfalfa Plant leaves) using Dynabeads mRNA purification kit. I have been following the protocol exactly the same but used 32ug (100ul) of RNA instead of 75ug and was eluting in 20ul . I used exactly the same proportion of reagents as mentioned in the protocol. Still i got very less yield ~ 17ng/ul. The concentration of RNA was 319.7ng/ul from which i was trying to extract mRNA. I was following the given protocol .. https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0015808_Dynabeads_mRNA_Purification_UG.pdf .. please let me know if anyone has some opinions on it... it would be really helpful :)
So from my reading you put in 32ug = 32,000ng of RNA, and you got out 17ng/uL * 20uL = 340ng of mRNA? That's just over 1% recovery which in my experience is pretty normal. Most total RNA samples contain 1-5% mRNA depending on the cell type and you'll never get back all of it, so honestly 1% is pretty good. What do you intend to do with the mRNA? 340ng of poly(A) selected mRNA is quite a lot and it should be plenty for most downstream workflows.
If you do want to try to optimise recovery, the first thing you should do is check your RNA integrity because poly(A) selection yield is extremely dependent on input sample quality. Do you have access to a BioAnalyzer or TapeStation to check the integrity of your input sample?
Laura Leighton ..That is a good point. About the calculation, i was not keeping in mind 340ng but instead 17ng/ul.
So, basically i need to validate miRNA cleavage on some of its target genes through 5' RACE for we needed mRNA and ligate with RNA adapter. I am planning to use thermofisher Generacer kit for this where it is told to use 7ul of mRNA.
I got confused with this statement 'Using amounts greater than 5 μg total RNA or 250 ng mRNA may require increasing the GeneRacer™ RNA Oligo.' So, i was expecting atleast 250ng.
We don't have agilant Bioanalzyser, I normally do gel electrophoresis and see through nanodrop the quality.
I am going to use this kit https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fgeneracer_man.pdf. Following from page 21, since in our case when miRNA cleaves the mRNA it will produce 5’ end monophosphate of the cleavage targets which binds to the RNA adaptor which is why Total or poly(A)+ RNA is directly ligated to the RNA adapter without any further enzymatic pretreatment.
Thank you so much :)
Do you have electrophoresis results for the total RNA input sample? If the RNA is partially degraded, then you'd expect to recover less mRNA, with the reduction in recovery proportional to the amount of degradation. (If the mRNA is damaged, the beads will only ever capture the 3'-most fragment which has the polyA tail, so you'll see a drop-off in recovered mRNA mass.) For this kind of experiment, differences in RNA integrity between samples/groups could really affect your conclusions, so if you can possibly get access to a bioanalyzer (even if you have to pay for a facility to run your samples) I personally think it would be very worth it.
With the kit you linked - "You may use either 1-5 μg total RNA or 50-250 ng mRNA with the GeneRacer kit". The maximum sample input volume is 7uL. 17ng/uL * 7uL = 119ng, so if you use 7uL of your existing sample you're well inside the optimum input range for this kit. If you concentrated your sample into 7uL you'd actually saturate the kit, plus it would require more manipulation of the RNA which could reduce its integrity, so if it were my experiment I'd just put in 7uL of the sample as-is.
Sameena Alam I have done 5' RACE and I will tell you that less input is usually better. Putting in too much template will suck down the primers early and you will only get a faint product. you could also do a nested RACE where the adapter primer and a 3' primer are used in first round of PCR, then gel purify that product and dilute 1:10, 1:100, 1:1000 and use in a second round of PCR with adapter primer and a 3' primer that is upstream of the first one. this will give a robust product that you can send for sequencing.
Laura Leighton , I have asked my supervisor about the Bioanalyzer - probably i can get access to it.
I have added the gel pic, the quality of the pic is not that good. I normally get sometimes some additional bands, can u also tell me what do they signify?
Julie Ann Dougherty .. i have couple of questions:
1. Can you tell me what kit u used for 5'RACE? I am planning to use the linked kit which is from Thermofisher Generacer https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fgeneracer_man.pdf but it is getting delayed in the shipment.
2. In the Reverse Transcribing mRNA step, since i need to do 5'RACE, i will be using Random primers?
3. What polymerase do u suggest in PCR, here they use Platinum® Taq
DNA Polymerase High Fidelity or PCR Setup with Platinum® Pfx DNA Polymerase but for this we need to add every component individually (dNTP and MgSO4. Superfi instead has everything included.. What do u suggest ?
Thank you
Sameena Alam I did this over 10 years ago and didn't use a kit, used "homemade" reagents. Generacer should be good though. using either the random primers or oligo(dT) should be fine. i was interested in only one target so i used a gene-specific primer. superscript3 and did perform RNase H treatment. using random primers might create multiple products for the same 5' end that you need so just keep that in mind. if your target is not too long and oligo(dT) would work then use that, or if you know your target mRNA and have an idea of the cleavage site then choose a downstream gene-specific primer for cDNA synthesis then the size of the product should also give you a ballpark idea of the cleavage site.
Polymerase should just be one to fit your needs, if you need one for a long product (>1kb) or a shorter one. I used an invitrogen hot start one. here is my pub on it.
Article Quantitative Analysis of Deadenylation-Independent mRNA Deca...
- Badges
- Science topic
- Similar topics
- Chemistry