I want to simultaneously isolate RNA and DNA from THE SAME Anopheles mosquito samples. I have been trying different variations of the TRIzol protocol (back extraction buffer, sodium citrate) and have had little success in recovering good gDNA. My 260/230 ratios have generally been poor (0.30-0.60) and the absorbance peak at 270 nm! What's more puzzling is that when I run the gDNA on the gel, I get no bands!
I know there are some kits out there that can be a better alternative but I was wondering if anyone can recommend a particular kit from experience?
How much nucleic acid are you recovering in the first place, pre-purification?
According to the nanodrop, from a single mosquito I get a range of 9-30 ng/ul in a total 20ul volume....though its hard to tell how accurate it is given the low 260/230 and the fact that there are no bands on the gel
How do you grind the cells to realese the nucleic acids? Do you macerated mechanically or by enzymatic digestion, or both? or only with the Trizol?. Maybe the problem is that you are not releasing completely all de the nucleic acids available or could be the opposite. You would be breaking the nucleic acids if your procedure is too harsh (I dont know). To isolate RNA, I usualy use the Promega "SV Total RNA isolation system" with tissue quantities as few as 10mg with good results. I hope this tips can help you.
Hi Danica,
we are doing some similar extractions with several triatomine bug tissues. Our initial yields were similar to yours, but after several months of adjustments we are now able to get values well above that (ranging between 40 and 100ng/ul).
We use a TRIzol protocol (our previous experience with two different commercial kits was much worse!!!) and I can tell you that the grinding of cuticular structures has been a critical step to increase yield, as well as the care taken to avoid RNAse action. This means avoiding using water that is not RNAse free. All reagents that you use should be taken care as to avoid RNAse contamination. The fact that you do not see a band might indicate that RNA is being degraded, but this is not a fact. We use RNAse away to clean dissection instruments and all the other stuff. Moreover, try to get access to a QBit method to evaluate RNA sample content. As far as we are experienced, the Nanodrop is not the best way to quantify (inconsistency has been common here and other people told us similar experiences). If you are afraid about degradation and want to evaluate that, then you will need to use a Bioanalyzer (you need a kit for that and it is not cheap, though). Also, do your best to eliminate TRIzol afterwards.
Good luck and do not hesitate in asking any other questions.
Marcelo
Some such type of error may occur in my sample also which was 290 nm present but band were not appear and one sample was only 29 nm but it show bands. its happen some time. repeat PCR with change temperature.
Dear Danica, try to use Chelex 5%. I use it for DNA extraction for bee legs.
In a well put the leg previously cutted with 100ul of Chelex 5% and 5 ul of Proteinase K.
Good Luck
Hi! See the following link it may be useful
A comparison of three methods for isolating RNA from mosquitos
http://www2.fiu.edu/~noriegaf/papers/Noriega-Wells%201993.pdf
Santosh Ippar asked about DNA isolation from mosquitos in the month of July. I gave the following links check
The following may be useful. Good Luck.
http://openwetware.org/wiki/Kafatos:genomic_DNA_extraction_from_individual_mosquitoes
http://www.mr4.org/Portals/3/Pdfs/Anopheles/3.12%2096%20well%20DNA%20extraction%20protocol%20v%201.pdf
http://journals.cambridge.org/action/displayAbstract;jsessionid=C8CC1C9FE6A515976E50C2AAB19AF07B.journals?fromPage=online&aid=763820
Isolation_of_genomic_DNA_from_mosquitoes_or_other_insects_using_the_QIAGEN®_Genomic-tip.pdf
I use Trizol for RNA extraction in my lab. First experiment, I have same problem with you. so, I just optimize PCR temperature (gradient temp PCR) and always using PCR master mix on cold condition (on ice) then resolve my problem. I hope can help you....
Hello, Danica, I suggest you to purchase a commercial kit for DNA isolation such as Qiagen DNeasy Blood & Tissue kit. When you isolate, please take several late staged larva or un-blood fed adults to grind, then finaly dissolve DNA in 100uL water or elution buffer, you can get good amount of DNA. Also when you measure the concentration with Nanodrop,the values of A260 and A280 as well as A230 do not make sense if your concentration is too low. For the RNA isolation, suggest purchase different kit from Qiagen. Best
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