Dear Experts,
I am working on RT-PCR analysis on arabidopsis genes. I notice that recently when i isolate RNA and convert it to cDNA, some sample give no/low amplification of housekeeping gene.
First of all, i isolate total seedling grown on MS plates using Qiazen qiazol lysis reagent. Finally i check on nanodrop and i get average 2000ng/ul RNA with nearly 2.0 of 260/280 ratio and 1.4 of 260/230 ratio. Then i convert 3ug-5ug of RNA to cDNA using invitrogen M-MLV reverse transcriptase and perform RT-PCR using UBQ10 gene as internal control to check my cDNA quality. Some sample gives a good amplification while some of it give a low/no ampification. I did realized of RNAse contamination issue thus i changed all compenent and run RNA directly on agarose gel on both samples with good/bad amplification. RNA bands are all visible and looks similar between both.
Do any of you have similar issues and know to solve it?Thank you very much for help!!
Are you house keeping (or reference genes) selected from relevant literature or did you do an experiment to determine which ones to use. When starting any gene expression or qPCR work you must all check for the appropriate reference genes and if possible investigate / select your own, preferably a panel of 2-3. You should check the MIQE guidelines (http://www.ncbi.nlm.nih.gov/pubmed/19246619).
RNase contamination can be difficult to get rid of as well. If possible you should check your RNA on a bioanalyser and not a denaturing agarose gel, as these can be incredibly tricky, especially if there is possible RNase contamination.
Also the nano drop ratio's are only an indicator and not that accurate. Any RNA extraction should be done using TRIZOL (for the fractionation) and then the upper aqueous phase used in a kit (such as the Ambion Purelink RNA extraction kit) to recover the RNA. This will get you the best quality RNA (Nuclease free as Trizol stops all proteins). RNA recovered this way will be of such high quality (as long as the rest of your prep is good), that it is good enough for array work or sequencing and will have a RIN (RNA integrity number) value of ~8-9 (max is 10).
Hope this helps, let us know how it works out!!
Thanks for the suggestions,
First of all, RNA isolation method and housekeeping gene i am using is working well as its being used by our lab for quite some times and validated by several publications. Initially i thought the problem could be on RNA itself but after i failed to get housekeeping amplification from pcr, i did reisolate the second set of samples but it still gives the same result as first one. Its quite weird since most of my samples give good amplification only some of it not.
As showed in attached gel picture, the only difference i could see from RNA loading on agarose gel is the band intensity. First three RNA samples i am loading here is the one without amplification whereas the forth one give good amplification. Number on each top band is the RNA concentration (ng/ul) i measured using nanodrop. i will try to play around with RNA concentration for cDNA synthesis. Usually we are using 5ug RNA and i will try as low as 1ug for conversion.
- Badges
- Science method