Hey, try using qRT-PCR reagents from Invitrogen, they have RT-qRT-PCR one step pre-mixes for low RNA yield and also for amplification of low-copy number genes...

Hi Yulia,

Best for qPCR is to run around 20 ng per reaction, but you can go down to 1 ng, which will likely be your case. It mostly depends on the level of expression of your genes of interest. A highly expressed gene may be detectable even with 1 ng, while other genes may not even show up with 20 ng.

A good trick is to increase the number of cycles, for instance run on 50 cycles instead of the usual 40. Although you'll have to be aware that this technique will also amplify the error on the qPCR and will increase the risk of amplifying non specific products (check carefully your non-template controls).

Good luck!

Dear Yulia, you should be careful with termination:

RT-PCR= Reverse Transcriptase-PCR

qPCR=quantitative (RealTime) PCR

Cheers, Nadine

Hi,

I think the best strategy depends on your aim. Do you already know which gene(s) you want to quantify? If you have one (or a few) candidate gene(s) only you may suggest a one-step qRT-PCR approach. It is even possible to make a multiplex one-step RT-PCR (only a few PCR cycles) and split the resulting RT-PCR products into singleplex qPCR reactions. You should take a look at some papers working on single-cell PCR (i.e. Peixoto A et al. Genome Research 2004)

Good luck!

Even if you have low RNA concentration, the most important thing is that you have pure RNA. Check your OD ratio 260/280 and make sure that it is between 1.8 and 2.2. You can now reverse transcribe your RNA into cDNA using High Capacity cDNA kit from ABI for example, there are other kits with other manufacturers. Don't forget to include an RT negative during RT reactions. Once you done with your RT which you started usually with 1ug RNA, you can assume that you have the same amount of cDNA at the end of the RT. You can dilute your cDNA and follow Nicholas instructions. Always include a housekeeping gene (GAPDH, 18s RNA, Beta actin, PPIA, ...) and an NTC to check for gDNA contamination and contamination of your water. Dissociation curve and efficiency of amplification between Target and housekeeping are very important to check before proceeding to plate run.

Good Luck

Following up on Nadine's comment:

Are you performing RT-qPCR ([one-step] reverse transcriptase quantitative PCR)? or reverse transcriptase PCR (therefore resolving products on a gel?).

But, assuming that these series of posts fall under the quantitative (real-time) PCR heading, I'll assume you mean RT-qPCR.

If so I think Nicolas Pillon and Norman Haefner's suggestions are right on.

Aorta must be a tissue with low RNA abundance (like brainstem and others)... So, this may always be the situation with aorta - and you have to use what you get.

When loading RT-qPCReactions, it is sometimes helpful to think in terms of how many cells' worth of RNA you are loading per reaction.

Given the estimate of 15-20 pg of total RNA per mammalian cell, 1 ng of total RNA per reaction means that you are loading 50 to 67 cells' worth of RNA per reaction; which should be fine if there are at least 5 copies of your target of interest present in each reaction. Assuming your samples are of high quality and high purity; Forge ahead~!

Hi, Yulia,

Don't know how you extract your RNA. I used to extract RNA from mouse aortic valves part to get 50-100ng/ul in total 10-15ul with high quality. And what's the volumn of your RNA solution? If it's relatively high, 100ul for example, you can concentrate it, and reverse all. Good luck!

Hii..

Probably, you would have got your solution from the above mentioned replies..One suggestion, while collecting mouse tissue you could snap freeze in liquid nitrogen and store it for overnight..Your RNA yield will increase and once you have good RNA yield, all your downstream processing becomes easier..

Keeping moving !!

How do you extract your RNA???

@Jack: Dear JAck, the termination qRT-PCR shouldn't be used anymore. The new and non-confusing termination is qPCR. Cheers, Nadine

Thanks everybody for your suggestions! I was using rotor homogenizer to disrupt thi tissue and then RNA easy kit from Qiagen. I'm going to precipitate my RNA to concentrate it and then proceed with RT and qPCR

You might consider using the RNeasy micro kit instead of the standard RNeasy kit. A colleague tested it and got significantly more RNA and higher quality from her samples when using the micro kit. RNeasy is fine if you have lots of sample/RNA in your sample. There is also the RNeasy plus micro kit, including DNAeliminator columns...not sure how well these perform...probably super excellent according to QIAgen ;-)

Other thing to consider: Run an agarose gel of your total RNA for quality control checkup. If your RNA is not degraded as, you should see the 28S and 18S bands. If you have access to the RIN as suggested by Jack in other topic, then you should be good to go. Again good Luck and always have your RNAse ZAP (AMBION, SIGMA...) to clean up centrifuge, your rotor, and your bench.

Hi,

There is a lot of useful suggestion here, another thing you can try to extract RNA with Trizol and use sonicator to disrupt the tissue.

Hey, try using qRT-PCR reagents from Invitrogen, they have RT-qRT-PCR one step pre-mixes for low RNA yield and also for amplification of low-copy number genes...

Hi,

instead of a rotor stator homogenizer, you could try fastprep cell lyser wich give better results in tissue lysis and homogenization

http://www.zymoresearch.com/devices-instruments/cell-disruptors/fastprepr-24-accessories

We had that problem for some tissues and went back to the old trizol/chloroform extraction. You can still do a DNAse treatment. Much higher yield. Used this protocol:

http://medicine.yale.edu/keck/ycga/Images/TRIZOLRNAIsolation_092107_tcm240-21453.pdf

I have had best luck with trizol/chloroform as well... always very good yield - as Jacqueline has just said. Also, if you maximize the aqueous layer you can recover at least 85% of it (or ~90% if being really careful to avoid the white gDNA-protein interface layer). OMNI TH has been the best homogenizer for us, but when doing 12 samples at a time, we use the rustic "Bullet Blender" -- a nearly Spartan device that pummels the tubes with a spinning roller-coaster like rail - it violently agitates 4 stainless steel ball bearings inside the tubes to macerate the tissue. Sounds horrible - very loud, but is successful (self-cooling as well). We hide the device inside of a vacant culture hood with the door fully closed to block out the hellish noise. If you want to frighten your kids next Halloween, you know what lab to visit now.

I have had the problem of low concentration RNA in the past too. You can easily solve the problem by using RNeasy mini kit instead of the Midi kit as suggested by Christoph Metzendorf. A few other things you might find helpful include using the recommended amount of tissue (when I used more I thought it would give me more RNA but ended up clogging the spin column), use a sterilized Scarpel blade for scraping -80 tissues to get fine samples for lysing and homogenizing last and most important, always do a second elution of the RNA. Most time I get a higher concentration of RNA in the second elution.

Hope this helps.

ps For qPCR I always have to dilute the RNA to 20ng/ul. (Beats the reason to want a high concentration in the first place)