Dear fellow researchers,
I am working on rat brain tissues. I need to extract RNA from here. The recommended RNA concentration is >150ng/ul (reading from Qubit). I am using Qiagen products and strictly adhere to their methods.
Thank you for your reply, my target tissue is obtain from tissue section. I am sure the is no fat components. It is kind of punching the tissue.
Try using manual extraction methods using such as Trizol or promega total RNA extraction kit (that will end up in getting a pellet) to get higher yield rather than spin columns.
Sounds like you are not lysing your tissue sufficiently. I've encountered similar problems with EBs in the past and I've had to mechanically mince them up before lysing the cells. I would then use Qiagen's QIAshredder columns to homogenise the cell lysate. Then you have a choice of different column based RNA extraction kit for various tissue types e.g. Fibrous tissue, Lipid Tissue etc. Hopefully this should increase your quantity and quality of RNA.
Dear all, I truly appreciate to all your replies. Let me clarify my source of tissue. My research is about hypertension. We are investigating the regulatory mechanism of this disease. As we are aware, there are certain regions in the brain that play pivotal role in regulating BP. For example, nucleus tractus solitarii (NTS) that act as cardio acceleratory and inhibitory centre.. Thus, I will harvest the brain, freeze them followed by sectioning using cryostat. I will identify this region with the help of stereotaxic thereafter punch it. I collect the punchers and these are my tissues which I need to extract the RNA. I perform my extraction using QIAzol; I have optimized this technique earlier to get the recommended ratio of A260/A280; and I am getting the ratio between 1.9 to 2.1.
I am actually pooling my samples for transcriptomic analysis. As I mentioned earlier the required concentration using Qubit is >150ng/ul. I am just performing nanodrop at the moment with my practise samples. I believe nanodrop should be showing higher compared to Bioanalyzer and Qubit flurometer? Correct me if I am wrong.
To add to the above suggestions, i will also suggest that you increase the volume of extraction reagent (whether trizol or lysis buffer) used in the lysis process. Low volumes of lysis buffer most at times results in low quantity of RNA extraction.
Low A260/A230 is mostly caused by contamination of your isolated RNA by residual organic solvents such as phenol. I will suggest you repeat the chloroform addition step twice to wash away most of the trizol after pipetting out your aqueous, clear supernatant.
Lastly, to get rid of the chloroform, washing the RNA pellet twice with ice cold 75% ethanol has been tried and tested to work in most cases.
All the best
Let's get this straight. Pure RNA has an A260/A280 of 2.1. You will see in many protocols that a value of 1.8-2.0 indicates that the RNA is pure. As Robert rightly said, low A260/A230 is solvent contamination and most of the time does not affect downstream applications. It also correlates with quantity so you will find that the more RNA you have then higher this value will be.
I would stick with Qiagen's columns rather than mess around with the old school Trizol and Chloroform.
Yes I agree with you, Sun Yung (Dr). When the yield of RNA is high the ratio of A260/A230 is high. I do agree with Robert (Dr) which it is solvent contamination. My counterparts at UK also mentioned that this will not affect the downstream application. I am thinking of dual elution.
Try adding more of Lysis solution and I would also ask you to take the reading in nanodrop after allowing your RNA to stabilize and dissolve uniformly in elution buffer
Correct me if I'm wrong, but my impression is that you're extracting RNA from very little material to begin with. You may just need to be pooling more samples as you've mentioned. If you're worried about inefficient extraction, you can use the columns as others have mentioned or add glycogen the the aqueous phase prior to isopropanol precipitation to act as an RNA carrier. As for contamination you can increase the volume, duration and the frequency of the ethanol wash. I too was getting low ratios using Trizol until I increased the volume of the ethanol wash to 1 mL rocking for 5 min x 2. From then on my 260/280 was always 1.95 and 260/230 was 2.30 (mind you I'd have concentrations in the ug/uL range)
Unless I missed something, I don't think we are clear what you're dealing with. Is your RNA concentration too low? If so, how low? It's strange (although not uncommon) to express the recommended amount of RNA in terms of concentration. >150ng/ul in what volume? 1 ul? 100 ul? It's always more useful to use the actual amount, e.g. 1 ug of RNA. It's possible to have enough RNA, but it's too diluted. In that case you can concentrate it. On the other hand, if you've got a very small volume of RNA, it may not matter that it's the right concentration. One good thing about Qiagen kit is that (at least until recently) they used water to elute RNA. Which means that you can easily concentrate it in the vacuum centrifuge.
As an independent endorsement, I've stopped using the Qiagen RNA extraction kit years ago. I much prefer the following kit from Norgen Biotek:
https://norgenbiotek.com/product/total-rna-purification-kit
It works just as well in terms of the yield / purity, but it doesn't involve any form of Phenol (including Trizol), the protocol is very fast and straightforward and it's about half the price of the Qiagen kit. I understand that you might be reluctant to change the kit/protocol mid-experiment. But I would recommend keeping it in mind for the future. As I said, I have no relationship to the company. I just really like their product...
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