As you can see there is no single answer..... the guanidinium-derived methods tend to work quite well (Trizol, RNAzol, etc) they are based on a guanidinium-thyocianate lysis (4M) and an acid-phenol extraction. The kits usually also lyse with guanidinium but then use the kit to purify the RNA without using acid phenol. You should try...

I have extracted RNA from whole blood using the Trizol method and then cleaned up the RNA at the end with the RNeasy kit from Qiagen. I usually get high quality RNA for cDNA conversion and very reliable RT-PCR data.

My colleagues use QIAamp from Qiagen as well and swear by it.

I m also using trizol method and it is good in quality as well as quantity aspect overall good results in real time pcr quantification so i think it is best 4 rt pcr purpose.

it depends on the sample amount. RNA isolation kits are nice when you have small samples but the fact that you have a mesh upon which the RNA and protein hold onto is somewhat a dirtier process. If you have a large sample go for a TRIzol extraction - it's much cleaner. If not, stick to the RNA extraction kit with columns.

Well the sample like I mentioned are generally fresh 5 ml peripheral blood or sometimes RNAlater stored buffy coat cells. What happened off late was that the mixing of stored samples in TRIzol wasnt good so yield was less. So I actually wanted to know whether the protocol used in TRIzol reagent guideline is sufficient or good to follow or a better one has to be used?

Whole blood is lysed with RBC lysis buffer and then trizol treatment ..thats one way else take out the buffy coat layer of WBCs and then add trizol to that. Which one gives better yield?

I think RNA isolation kit, good quality but lower amount

I recommend tri-reagent from MRC it works better htan trizol, If you use it, try to use a smaller volume and double the chloroform. Say 100 to 200 ul/ million PBMCs it will help in the precipitation step.

If you can remove the RBC before lysis using ficoll, or a lysing reagent, The hemoglobin may interfere with your later reactions.

Good luck with your reserch

You can try RNA extraction kit pruduct by QIAGEN; if you have more than 500.000 cells you should use MicroKit RNA extraction otherwise try MiniKit RNA extraction.

Extraction by Trizole is a good method when you work with more than 5 million of cells.

You can try RNA extraction kit pruduct by QIAGEN; if you have more than 500.000 cells you should use MicroKit RNA extraction otherwise try MiniKit RNA extraction.

Extraction by Trizole is a good method when you work with more than 5 million of cells.

You can try RNA extraction kit pruduct by QIAGEN; if you have more than 500.000 cells you should use MicroKit RNA extraction otherwise try MiniKit RNA extraction.

Extraction by Trizole is a good method when you work with more than 5 million of cells.

I think it depends on how frequent you have to extract it from blood, how many experiments you need to do out of each sample, and whether it is your routine work? I have used both, infrequently, for the past several years for my work. To me the extraction kit is very convenient whereas Trizol method is more time consuming. After several experiments by Trizol extraction I switched to extraction kit. Since I am not routinely extract RNA so the purity from the kit is always better than using Trizol method (personal experience). All my students use the kit since they all feel it is easier to follow the manual for the extraction and they can complete the extraction in less than half of a day.

Dr. Teng, I am actually coordinating with another MD student and he will have to do it routinely as in many processing at the same time. We in the lab follow kit too but again too many samples we go for trizol. Actually we have been getting RNA good with both ways and its not so routine. Only for this project we might go for routine analysis from Whole blood (fresh and frozen cells) as well as tissue (brain tissues frozen in RNA later). So this protocol takes half a day. for microarray we always used RANeasy kit.

Ofcourse if fund is not a problem kit is the best one. Trizol also works well. For that you have to collect the blood in vacut tube filled up with RNA Later.

We normally use the mRNA isolation kit from blood/bone marrow from Roche. It works very nice for us, we usually use RPL32 and RPS18 as reference genes and the CP (or CT) values for these genes are around 15 to 17 for 5ml blood. We have done extractions from blood stored one year at -80 in stabilization buffer (RNA/DNA Stabilization Reagent for Blood/Bone Marrow also from Roche) and they have worked very nice. We use the SuperscriptIII from lifetech for cDNA.

Hi Kalpita: Thanks for your response. Kit will save you time but costly. But, consider to label multiple tubes and not making mistake then kit mabe easier to do your experiment. If your student is a very hard working one, and cautious enough, then feel free to use Trizol method. I use kit simply because my students have a lot of commitments and requirements for lectures and clinical responsibilities if the extraction takes more than a few hours then they will end up as my work. I guess you already have the answer.

I agree with alfredo about qIAGEN RNA extraction kit by using column method

Well I think trizol is good option to extract good quality total RNA as in qPCR you also need smaller ribosomal subunits sometimes along with major subunits, which can easily be extracted from trizol reagent. I'm using trizol from life technologies for extraction of total RNA from whole blood and its works well in samples of around 10-12 ml and I think it is a good option even for small sample quantity. Make sure you process all samples in refrigerated centrifuge at 4 degrees and avoid Rnase contamination and also aliquot the extracted RNA so that it can be prevented from repeated thawing from -80 degrees, so that you can analyze the quality of RNA on gel and nanodrop without affecting the major stock which can be used further. Kit is also a good option try for roche extraction kit. And about holding stage try to store the pellet of leukocytes obtained after centrifugation of layer above histopaque. I think this will solve your problem.

Thanks Samarjeet. Well feel trizol and chlorofom step, some shearing is happening whcih is why, the quantity might be fine but gel and nanodrop peaks aren't sharp at the right OD.

Thanks Dr. Teng, my colleague is new to molecular biology, basically a clinician hence I am coordinating to help him. Ya samples might be little large but I take care. Right now we are standardizing and we use ABI taqman assays, there instrument and even the cDNA synthesis kit is from them. The only thing is to get non sheared good quality RNA.

Hi, I was recently working on extracting a good quality RNA from total mouse blood.

So I ended up using the ABI's Mouse Ribopure kit.

At the moment of obtaining the sample, you mix it with RNAlater and can process it months later. As you can see from the attached image, the quality is exceptional!

That kits extracts the small RNAs also. The only drawback is the presence of genomic DNA, and a DNAse treatment should work.

I am currenly making trials to check which method of DNA removal would allow me to have the small RNA still present.

I agree with Petar that DNAse treatment may be necessary to avoid gDNA interference for the RNA extraction. I am not specific to any extraction kit except I only tried Qiagen kits before (not attempting to promote it) since it was the one my lab used. As a clinician like to do some bench research with limited microbiology traning and learn how to do it all by myselves from scratch the kit method is easiler for me than the Trizol extraction. At least I do not need to worry too much about calling away from bench for unexpected sick patients.

Hi Peter, You know same kind of exceptional bands I have gotten with trizol reagent itself with almost nil gDNA contamination and a blotch of small RNA too. My problem lies now in the extraction procedure. The mixing of the pellet to the trizol or mixing of whole blood post centrifugation to the reagent. Will see if this doesnot work out in human blood and brain tissues for which I am even more worried, I will switch to Kit RNAeasy but people say Ambion Kits are good in removing most of the gDNA contamination but yes I do DNase treatment.

Hi Kalpita, for the brain I will go for RNAeasy lipid kit, or I would take the aqueous phase from TriZol and will process it with Rnaeasy kit (or similar), which allows for on-column DNAse digestion (but you will lose the small RNAs).

We use this mixed protocol for adipose tissue in our lab and as an additional step I always precipitate with sodium acetate/ethanol overnight. This assures that all samples have A260/A280 higher than 2.1 and A260/A230 higher than 2.2 .

HI Kalpita. I think (As i did)

Highpure RNA isolation kit belongs to Roche company is working well, i think is the best one because it contain DNase treatment step by itself.

Thanks all. can I ask my question now in a modified way saying that How can you get better RNA from stored blood samples? as in where the lymphocyte pellet has become very hard. I am facing problem in making a homogenous mixture of such samples.

for me i use Qiagen kit ( special for Blood extraction RNA) i have a good quality and quantity

good luck

Hi Kalpita, I have used PAXgene blood exraction kit for human samples. I am not sure is there is smaller version for mouse, but it worked just fine. It has a sample preservation solution in collection tubes. We were getting good quality and more than enough RNA to do bunch of analysis. Take a look at the link.

Best,

Vlado

http://www.qiagen.com/Products/RnaStabilizationPurification/DSP/PaxGeneBloodRnaKitIVD.aspx

RNA extraction KIT is easy but expensive

Trizol i think is good

The quality will depend on the sample storage methodology and the life span of the stored sample. However, methods which rely on silica columns will give a good yield and higher quality of your preparation.

Hi. I personally like the Ambion Ribopure kit. The RNA quality and purity has been unmatched. I combine the kit together with phase lock gel from 5 prime (for the phase separation step). The only drawback, high per sample cost.

In the past I have used Trizol and Qiagen RNeasy with good results. I agree with previous comments about sample quality influencing RNA quality.

there is a discussion thread which might be relevant to your lymphocyte pellet question happening here: https://lists.purdue.edu/pipermail/cytometry/2008-April/034897.html

Thanks Jacqueline...its helpful. Ya my colleagues in his clinical setting doesn't take enough care in storing cells I guess but anyway I will figure out which method to go for. Combination of Trizol and kit seems good. Will have to try that and see.

I think and our personal experience about it and i m evaluate both of them methods TRIZOL and Roche Kits, and Invitrogen kit for RNA isolation efficiency of band pattern of these methods results are TRIZOL is excelllent rather than commmercial kits. And if you are not very sure for contamination then use DEPC treated vials.

I have done a lot of isolations none of the above are useful for qPCR the magnetic system is far more superior if that is an option and available where you work would be ideal.

Dr M A Choudhry

As Jacqueline mentioned above, I have also been extremely pleased with the Life Technologies/Ambion RiboPure Blood kit, which combines organic extraction with downstream spin columns. I followed this with a globin-removal step and DNase-treatment. I have had excellent results on Affy chips using this kit.

We did collect the blood directly into the provided RNALater tubes, and I can confirm that blood samples were stable for up to three days prior to RNA extraction.

I have used the Paxgene tubes in the past, but found my results to be better with the RiboPure blood kit.

Trizol will work if you will use the RNA for qPCR. For applications requiring cleaner RNA, such as array, you might need to do an extra purification step afterwards (phenol-chloroform). I use regular Trizol which is not optimized for blood, but you have to use alot of it (1:10 ratio to whole blood). Trizol LS is more concentrated so it could be advisable to get that. You could also remove the RBC before using Trizol.

I always go for Total RNA miniprep Kit (red filtrate) by Sigma which gives a better yield. The foremost imp thing is Lymphocyte separation, if possible go for cell counting then for RNA isolation (take the readings immediately using picodrop and store the samples immediately in -20 feasible -80 C), cDNA synthesis and qPCR.

Instead of trying with 2-step PCR also try with 1-Step PCR it may work.

RNeasy mini Kit used in combination with QIAshredder and DNase I should be fine.

The question would be to useTrizol as a less expensive way to get RNA for qPCR? If the cost doesn't matter, I would definitely go with a kit aswell (non-toxic!). However for most applications, Trizol will do just fine.

Something that you should keep in mind is that when someone extracts RNA, his/her manipulations should be quick at the first steps when the cells are lysed. This is because at that step all the enzymes that may digest RNA and belong to the cells, are released and to my opinion this is the most critical point that affects the quality of the RNA extracted.

If you need at least 1 mcg of total RNA Trizol is the best but you should be careful with the pellet of mononuclear cells not to have any red cells contamination, otherwise your cDNA synthesis would not be perfect.

In my experience, we used Trizol and phenol-chloroform for isolation of RNA for microarray applications and found a disturbingly high amount of phenol present in the final product. Obviously, this is a case by case basis but if purity is top on your list I recommend going with a kit! I have been using MoBio Lab's RNA isolation kits for years and they have never let me down. Alternatively, I have also tried the RiboPure kit and its also a great alternative.

Hello Kalpita, I just found a relevant technical paper about the topic :

Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

http://www.biomedcentral.com/1472-6750/12/33/about#citations

Hi, Peter

thanks for the paper. Its a nice one.

kalpita

I think both of kit and trizol are good choices for rna extraction

but the kit is more efficient

Hi Kalpita,

TRIzol and RNA Isolation kits both are good, I use TRIzol method and I generally get very good amount of RNA for cDNA synthesis and as well as Q PCR. But since your sample is clinical so it would be better if you use RNA isolation kits.

I agree with Klar and Sandeep. Well, TRI/Trizol are economical and a bit of tweaks and expertise is needed. Whereas, with the RNA isolation kit, just some common sense is required with the expence of some money. Also I feel that the samples isolated with Trizol/RNA kits would be 3:1 in number (as with the costs) and the Isolation by kits would save a lot of time with less volume of your isolates.

If you intend to detect both mRNA and sRNAs I would suggest you to go for Trizol. In my hands it proved to be more realible for both RNA sizes.

In my opinion you can use whatever you want, I have used isothiocianate -guanidine (old method), Trizol and Ambion kits, whith the same results, using kits you save time but not money. But recently I have used trizol to isolate mononuclear cells to test the expression of Ia cytoline. with good results.

It's depend on your goal. But, I think the RNA isolation kits more efficient and make ensure that your results would be better than using TRIzol.

I would say both are good. If you are looking for high quality total RNA preparation with guaranteed successful downstream applications and you have some spare money I would advise you to go with the kits such as RNAeasy kits from Qiagen. If you have a reasonable expertise and looking for a big yield and you have a delicate skills in the purification of aqeuous phase containing your RNA from interphase (containg contaminating proteins) and lower organic phase (containing contaminating gDNA) I would go for TRIzol (Ambion). Bests

Hi Kalpita

according to my sresaearch which had included many times extracting RNA ı used TRİzol which is more efficient than all kit. ıf you make a research comparing the efficiency between kit vs. TRIzol; TRİzol is the BEST !!!!

As you can see there is no single answer..... the guanidinium-derived methods tend to work quite well (Trizol, RNAzol, etc) they are based on a guanidinium-thyocianate lysis (4M) and an acid-phenol extraction. The kits usually also lyse with guanidinium but then use the kit to purify the RNA without using acid phenol. You should try...

For RNA from blood, we first isolate buffy coat (0.2mL), store the samples in 1 mL TRIZOL.TRIPURE etc. (samples are stable for at least 1 year) and then proceed with normal protocol, then we alway perform DNAse treatment followed by another TRIZOL (0.75mL) isolation.

I have Tried Trizol and other kits. Quality is good in both, but YIELD is MUCH better with Trizol. It is a bit more laborious, yes, but is more economical, and students learn a bit better the concepts behind the RNA isolation than when they use the kitchen recipes coming with kits.

The best RNA extraction protocol ever was optimized by Chang, 1993 to get RNA from pine trees. it is CTAB-based and most other protocols have been optimized from it to suit particular tissues etc

Agree with you that Trizol (in my case Tripure) is probably the most efficient method, especially when working with tissues. Blood cells are slightly complicated, and I always had a kind of not genomic or broken DNA contamination, which I never had before, so I was thinking to change the method. What do you think?

Hi everybody, I am a bit confused about the strategy I should adopt in the following case:- Collect HPBL, Isolate Lymphocytes using Fycoll, distribute in experimental dishes, give treatment for a few hours at 37 deg C, then set culture using phytohematoglutinin say for 24hrs, collect and suspend in RNA later, isolate RNA using RNAesay, and then finally quantitate to find that nothing is found....I am really confused what went wrong....kindly suggest. Also tell what could be the minimum amount of whole blood that can be used for proper yield in this kind of expt.

Regards

Nitin

We routinely use PAXgene tubes, they are quite expensive (about £7 per tube) BUT they are excellent if you have lots of different research nurses/doctors taking the blood. They are excellent for maintaining RNA stability after blood collection. As they are clinical trial samples I would definately recommend PAXgene tubes we have had great success we RNA quality used for gene array work using these tubes.

Hi, I've been using below protocol satisfactorily.

Fresh 3-4 ml EDTA or heparinized Blood.

Isolate lymphocyte using  BD Vacutainer® CPT Tube .

Wash 2-3 times with cold PBS with 5000 rpm centrifuge for 1min in 2ml tube at 4C.

Then centrifuged to pellet.

Isolate Total RNA with Trizol Reagent from the Lymphocyte pellet.

Take care to remove washing 70% Ethanol.

The washing step is very important to remove guanidine that is potent inhibitor of enzymatic process, but there is high risk to loss the RNA pellet.

About 1 - 3 ug / ml Blood can be obtained with this protocol.

Hi, I am agree with Jaqueline Lane and García Sanz, first,  if you dont have money , try to use trizol, it is very simple and  work well to islote RNA, and if you ,have money, it is better to use a kit for example Ambion, especially when you have to process a lot of samples and it save time, try to use also the Paxgene tubes 

Trizol would be better for initial stages. For ultra pure RNA for qPCR and to be free from gDNA, QIAmp(Qiagen) or Purelink (Thermo) are also good in terms of yield obtained. I experienced better yields with columns than organic extraction methods (like Trizol and GTC)

Would there be a lab/researcher interested to extract RNA from lymphocytes (from a blood sample)? If so, please get in touch with me.

I use trizol method ....once u add trizol... u can process later...if stored at -80 C...kit i guess u have to finish...entire protocol same day... n costly too...

Trizol method u need trizol..RBC lysis buffer, chloroform, isopropanol, ethanol..n ur done....

let me know i f u need protocol

Virendra V. Panpatil  .I  also need the Trizol protocol for the RNA isolation from the whole blood. Thanks

Virendra V. Panpatil . I also would like the protocol for RNA isolation from whole blood.

Thanks

Virendra V. Panpatil Can I have the protocol for RNA isolation from whole blood. Many thanks in advance

Using epicentre kit... not doing manually