I used Trizol reagent and isolate both RNA and Protein from frozen spinal cord tissue. I got good results.

Please see the attachment.

Good Luck!

Hi Elham, If you are using RNeasy mini kit to extract RNA you can use this protocol.

http://www.qiagen.com/knowledge-and-support/resource-center/resource-download.aspx?id=7b79fe96-71a3-467f-aa8b-767a9de6f200

As long as you use Trizol, you will have your RNA on the upper aqueous phase, your protein in the middle interphase and your DNA at the bottom.

Hi Elham,

Machery & Nagel have a Kit were you can extraxt RNA and protein from the same tissue.

Dear Mohamed Mahdy , really thanks. so helpful

Dear Joon Shim, I've already used Trizol Reagent for RNA and Protein extraction, but faced two problems. The purity of extracted RNA is very low and the extracted protein was not suitable for Western blot

Dear Pejman Rahimian, you're right but I could not used that Protein for western and the purity of extracted RNA was very low. But the RNA concentration was good. I also used kit, but the RNA concentration deceased so much, could not run it on gel

Thanks dear Petra Unger. Have you ever used this kit? the feature which I read through net was good, related to my question

Thanks dear Hardik Patel. Kit didn't work for me. Got very low concentration

Hi Elham, I have used the kit, but only for frozen skin. It works good.

I used this kit with B cell and I could see the proteins by WB

http://www.qiagen.com/products/catalog/sample-technologies/rna-sample-technologies/dna-rna-protein/allprep-rnaprotein-kit

I recommend Trizol. Make sure you resuspend your frozen tissue well in 1 ml Trizol. You may consider using the ceramic beads from Roche (MagNa Lyser Green Beads)/ Lyser Instrument (homogenizer) or any traditional homogenizer (without the beads) to homogenize your sample. The key point to have good RNA/DNA/protein yield is to make sure your sample is completely dispersed. You can get high quality RNA fraction (400ul) as long as you do not pipet as much as possible especially near the interface. Be careful not to disturb the interface either. Follow the protocol. However, be aware that the protein fraction is not easy to solublize, some time you need to incubate the sample in 1% SDS 50 C for hours. Anyway, once the protein samples are in SDS, they are denatured and are only good for Western.

Good Luck!

Hsin-Pai

Through Trizol reagent (Invitrogen) you can isolate both RNA, DNA and Protein. From upper aqueous phase, RNA/DNA both can be recovered either by using Isopropanol or ethanol as precipitating agent respectively. You can get protein from middle interphase.

QIAGEN Allprep (cloumn based) has always worked well for me either the RNA/DNA kit or the RNA/Protein extraction kit, it is just a bit expensive.

I have used this kit for muscles and got good results:

Good luck

QIAGEN product:

The AllPrep DNA/RNA/Protein Mini Kit standardizes sample preparation for systems biology. DNA, RNA, and protein are all prepared from the same source, eliminating the variation inherent in preparing these analytes from different samples.There is no need to split the sample into 3 prior to purification, allowing maximum recovery of DNA, RNA, and protein. In addition, no organic solvents such as phenol and acetone are required.

Please check this paper

Hope it will work fine as we are using this regularly.

Hello. We use Trizol method for tissue samples and usually we extract RNA and proteins and both of them are ok for RT-PCR and western blot, respectively. We have observed that when using a kit, RNA concentration is lower than when using Trizol so it is up to you or whether you can easily obtain more tissue sample (in our case we work with human samples, so we prefer to use Trizol although it can be a longer process)

On the other hand, you can improve the Trizol method by substituing the chloroform by phenol:chloroform:isoamyl alcohol (25:24:1). I advise you to buy it instead of making it. Also you can wash twice with 70% ethanol as the purity will be better.

If in the future you work with cells, we have seen that it is better if you freeze them in Trizol (avoid long periods of time).

I hope it helps.

I used Trizol reagent and isolate both RNA and Protein from frozen spinal cord tissue. I got good results.

Please see the attachment.

Good Luck!

Thanks dear Petra Ungerand Laura Garcia-Exposito

Thanks dear Hsin-Pai Li , but how was the purity of samples when you used the Trizol? I've already used it but the purity os my samples were very very low, less than 1.0 like 0.75 or 0.5

really thanks dear Anuradha Singh, Stella Tran, Mohammad Khan and Ravinder Kumar for your help. I will do them as you all mentions

Thanks dear Celia Escudero-Hernández, yeah you're right. When using the trizol reagent the concentration will be high but unfortunately the purity of product is not. This the problem? I used it by trizol and chlorofom and wash my product my Ethanol 70%, even I tried by absolute ethanol, but the purity still is very low. How you overcome this problem?

Thanks dear Samar Ghorbanpoor . I exactly fllow this protocol before, but as I mentioned before I saw the good concentration but purity was very low. RNA was contaminated by DNA and Protein

Hello,

I use RNeasy Mini Kit for RNA extraction, my RNA very pure and in good quantities although I use very small amount. You have to choice the right kit for you tissue. Western blot I homogenise the tissue after ground it down in liquid nitrogen.

Something that one of the labs over here is practicing. They use one hemisphere for mRNA study (RNA extraction) and the other hemisphere for western blotting (protein extraction). Saves using twice as animals.

Elham,

I have used the Trizol extraction method to obtain RNA and protein from frozen rodent PAG with excellent purity. Colleagues of mine have been successful with much smaller regions, such as nucleus accumbens. If you are concerned about the purity of your RNA, you can try leaving a small amount of the aqueous RNA layer (following the chloroform+spin step) to try to prevent contamination. I hope this helps. http://tools.lifetechnologies.com/content/sfs/manuals/trizol_reagent.pdf

Dear Petra Unger, do you have any related ISI paper which is related to Machery & Nagel kit for RNA extraction? I searched but could not find. Thanks

Thanks dear Akrem Abdulsid. Yeah I ordered for it

Thanks for your comment dear Mohammed Zubair Rizwan

Thanks dear Zoë H Brett. Yeah I've tried it but still the purity 260/230 is low. I have to think about it or change my extraction method

Hello Elham you can use easily miRvana PARIS.. i use it routinely for RNA and Protein extraction from frozen liver and plasma samples

Hi,

I have heard from the Company representative which visit our Institute that kits are available which can be used to isolate DNA, RNA and possibly proteins by using single kit. I will confirm and update you accordingly meanwhile please look for different companies kit manuals for the same.

Regards,

You can use trizol reagent but after phase separation using chloroform step keep both phases one for RNA and one for Protein and make forward them to obtain pure RNA or Protein

good luck

Dear Elham, use Trizol kit and just follow its own protokols. Its a good and in the mean time an easy-to-use kit.

Best Wishes

Dear Elham,

The Trizol protocol could be a good solution for your problem. All the steps are described.

Best regards

We are using trizol to isolate RNA . But  in my experience the guanidine method is the best to get  high quality intact RNA. Yes there are several kits in market as mentioned in the above answers.But try the guanidine method. 

Thanks all for your kind help for this issue. But I have one question here. You all that used Trizol reagent, did you check the protein for protein essay like Western Blot or  Mass spectrometry??? How you use it?? by which protocol?? Thanks again

We use to quantify the proteinsby Bradford-based assays. We have experience with DC™ Protein Assay and Protein Assay Dye Reagent Concentrate, both from Bio-Rad (the second one is faster). There are other methods available or you can even measure them using NanoDrop, but that quantification is not advisable for proteins.

After that, we perform western blot by loading 8microg of protein per well. To transfer the proteins to the membrane we prefer the wet method. For blocking you can do it with Tween20, BSA or FBS. Generally, the better one uses to be the serum of the animal where your secondary antibody was made. So, if your secondary antibody was made in goat, use 4% goat serum in TBST. You can prepare TBS 10x (0.5 M Tris, 1.5 M NaCl, pH: 7.5) and add Tween20 at 0.1% to the diluted 1x solution to make TBST working solution. We block for 1h at RT, then incubate with primary antibody at the recommended concentration in blocking solution (overnight, 4 ºC). The following day, wash 3 times in TBST and incubate with secondary antibody for 1 h at RT (diluted in 5% milk without fat). After washing, we reveal with Supersignal WB susbtrate. For relative quantification we use VersaDoc machine, but for qualitative purposes, we just use X-ray films.

If you want to check the proteins in general instead of a specific one, you can load a gel and dye it with coomasie blue. If you are going to follow with western blot anyway, you can dye the membrane with pounceau red. This one can be easily washed out with TBST before the blocking step.

I hope it helps.

Thanks dear Celia for your comment. But I mean in Trizol-base methods, how you isolate the protein from the Trizol / Qiazol reagent (phenol/ethanol supernatant)  for western blot , after you extracted RNA?

I know by the cold Acetone and guanidine hydrochloride (GuHCl) in 95% ethanol, we can precipitate  proteins which is present in the Trizol / Qiazol reagent. I would like to make sure this precipitated protein can be used for down approaches such as western blot or mass spectrometer? Is there any alternative reagent that could be used instead? I'm looking for the best and most convenient method for protein isolation from Trizol / Qiazol.

Ok. I'm sorry. I only have experience in western blot, so I am not sure if what we do is the best for mass spectrometry. In my lab we use the GuHCl method to obtain the proteins and continue as I described earlier. 

Really thanks dear Celia. So you mean you already used Trizol to extract RNA and protein from your tissue samples. Then use GuHCL to precipitate protein and then use this protein for Western blot?? Right??

The isolated protein is a denatured-protein, so is it ok for western blot? sorry I really worry for it and ask you for my confirmation.

Thanks alot

Yes, that's right. With Trizol we extract RNA for RT-PCR and proteins for Western blot.

Thanks alot dear Celia for your time

Dear There,

In my experience, i have tried to extract total RNA by TRIZOL or total Easy Blue at phase separation, followed by RNeasy cleanup (Quiagen) and get high yield and purified RNA. We can keep lower phase for DNA and protein purification at -80 oC later as Trizol's guidline. I hope that help :)

I want to ask another question. If we use TRIZOL for protein extraction from a sample, I want to know we can assay enzyme activity or not? 

Dear Do Lung Huynh, thanks for your suggestion. It will be perfect to use protein and RNA in a same time. 

Dear Babak Moradi, Thanks for your question. I don have any experience using TRIZOl for enzyme activity. I hope somebody can answer your question which will be useful for future experience of us. 

 Hey Elham, I just saw your question now and I am trying to do the same thing. Considering its 3 years later I was wondering if you had any success with seperating protein and RNA from the same tissue from the striatum and substantia nigra. Would you be able to tell me how you did it? Thanks

Hi dear Peggy. Actually, I used the RNeasy Plus Universal Mini kit from Qiagen to extract RNA and protein together. I got good quality of RNA  and kept the remaining protein in -80 for WB. However, I have not used the protein yet as my research was finished. Good luck

Hi all,

I am using only a segment of spinal cord and as the amount of tissue is smaller,I only used 500microgrm of trizol for homogenisation.The yield of RNA is too low,say less than 200ng and I am not able to see the pellet too after centrifugation.

Can anyone help me with this.Thanks!

Dear Nia, Good day.

Could you plz tell me how much the amount of your tissue?

I am afraid whether you have sufficient amount of tissue. Otherwise, you can grind the tissue in liquid nitrogen pre-chilled Mortar and pestle and divide the powder portion for RNA and protein separation. You should collect the powdered tissue in Trisol (for RNA) and protein lysis buffer (for protein) and proceed for further extraction !