Hello Fellow researchers !
I am doing RLM 5'RACE to validate mirRNA cleavage on it's target genes using GeneRacer kit (Thermo Fisher Scientific). After when purified mRNA using Dynabeads where i used ~32ug of RNA and yielded 17ng/ul for 20ul of volume. After searching i found it is potentially more than 1% of of my original RNA (~32) which i moved ahead with ligation step. I followed this link from page 22 https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fgeneracer_man.pdf and once i finished i ran 1ug of ligated mRNA with 1 ul of 6X loading dye and 2ul of water. I don't see anything on the gel except my ladder, is it normal? I was wondering if SYBR stain is good to stain mRNA or since this time i diluted mRNA to run on the gel and it was pretty less amount of mRNA which is why i did not see anything.
it would be really helpful to get your comments :)
Thank you
It is very dificult to check what happened after RT or ligation of a complexe mixture ... if you don't have used radioactive tracer... cDNA are in low quantity of different size... PCR will be the answer; if you don't have the expected PCR product then you will have to check your procedure with the controls in the kit. The main pb being generaly the qualtity of your RNA.
Didier Poncet , Thank you so much for your reply. I also re ran the same ligated mRNA with 3ul of volume instead of 1 and without diluting it. There i kind of got light band at around 100bp, I don' understand if it degraded or not? I have attached the gel pic so you can understand what am trying to say. The first lane is a ladder of 1000bp and last lane is the ligated mRNA with an arrow. Please ignore other lanes.
I have also checked my RNA quality, it was in good ratios. However, it is a good suggestion to check with controls in the kit.
Sameena, did you expect such a small product? (it does not look fine for me...) did you make the PCR? (if it is OK you should get a clear well defined band; do you have an idea of the size of the final product you could expect?
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