I have to isolate total RNA from needle biopsy tissue samples for the purpose of microRNA profiling by using next generation sequencing. I have purchased Ambion mirvana kits but after repeated attempts (I have used 6 different tissue samples so far), I am unable to obtain a good yield and OD values. The maximum yield I am getting from 4mg tissue is no more than 70-120ng/ul when dissolved in 50ul of nuclease free water and 260/230 value is always from 0.75 to 1.8. I have tried different steps to optimize yield from mirvana kit but with no success. So anyone who has experience with this kit please share your protocol so I could possibly improve total RNA from my samples without wasting anymore samples. These samples have been stored in RNAlater and there is no degradation as measured by Bioanalyzer (RIN>8).
Dear Emanuel,
Thankyou for your reply!!!.. Unfortunately it is not possible to get more than 6mg tissue when doing needle biopsy. Right now I am doing elution with 50ul nuclease free water. At first I tried with 100ul elution buffer but yield was too low per ul that I decided to try 50ul. I have to manage atleast 200ng/ul and a total of 20ug minimum for NGS. Don't know if its due to ratio of chemicals being used or something else =(. In your opinion, what could be the reason for this too low 260/230 ratio? Could it be possibly due to use of nuclease free water rather elution buffer? I am using nuclease free water just in case to avoid any issue with EDTA in elution buffer for NGS library preparation and later on real time PCR...
Emanuel,
I have attached the steps that I am currently performing...
Also, When I performed RNA extraction using mirvana kit for the first time, I used 1ml lysis solution and according to that other reagents but still there was no difference i.e., just 48ng/ul and 260/230 = 1.8.....In another attempt, I divided same tissue into two equal pieces by weight and extracted RNA with mirvana kit and TRIZOL. In case of TRIZOL, I obtained 250ng/ul in 50ul water from 3mg tissue (260/230=1.7) whereas 110ng/ul from mirvana kit (260/230=0.75)....
Hmmm I don't think there is issue of high or low content in my tissue samples. As I mentioned, I have managed to get even upto 550ng/ul from same amount of tissue (3-4mg) although each time this was with TRIZOL....Could you please tell me the condition of phases you get after centrifugation? When I finish up with centrifugation, there middle phase is just a thin line that it hardly distinguish aqueous, middle and lower phase. Most of the time it just appears as such that there are two phases only....unlike the case seen in TRIZOL where there is very prominent separation of three phases...I have tried by giving centrifugation more time (upto 15min) but this doesn't seem to give any better separation of phases. So, is it same in your case too?
Dear Emanuel
Thank you for your reply. Since you have done NGS, what do you say how much influence can a low 260/230 value have on expression profiling? Because somehow I am still getting 280/230 = 1.9 - 2.0 for same samples.
We used the Norgen kit for liver biopsy (5 mg of tissue) and we obtained quite good results. I do not know however, if the final amount is enough for your NGS protocol. Which is the amount of RNA you need?
Andrea the requirement is 200ng/ul and atleast 20ug in total....
Andrea could you please tell me how much RNA/ul , ng did you obtain from 5mg when using Norgen kits and in dissolved in how much water/buffer?
I have to check in my lab book. I will let you know next week. However, the final volume is 50 µl and if you need 20 µg of total RNA you should reach at least a concentration of 400 ng/µl that is rather difficult to obtain starting with this low amount of tissue. I will let you know
Another query....In column based kits, there are always two protocols. one is Total RNA isolation and other is enrichment of small RNA. So, in my case where I have to profile miRNA via next generation sequencing, RNA should be isolated as Total RNA or by the enrichment of small RNA method??
Azeem,
In the experience of my lab, Qiagen kits such as miReasy, provide greater yield of RNAs than the mirVana kit.
Considering the question about to work with total RNA samples or enriched samples, I believe that the best choice is not to enrich. During the enrichment for small RNAs you could lose some endogenous genes that would compromise your validation step, e.g. in a real time PCR experiment.
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