RNA extraction from blood - (Dec/13/2005 )

I think this is the right forum for this question.

My boss wants me to extract RNA from blood to do real time pcr on. Since this is present only in the wbc, that is what we are going after.

Two questions:
How much blood should I pull from the animals to extract a reasonable (30-60ug) amount of total RNA?

What system/kit/methodolgy would be best to use for this.

I've extracted a lot of RNA over the years, but never from blood.

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Yeah, well.... thanks for your help gang.

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ok for your 2 questions i'm not an expert as i don't work on blood...
but :

starting with 5.10^6 cells should do the job

i use trizol for rna extraction.
if your sample is frozen, preheat at 50° and add directly and resuspend the pellet. Accelerates the lysis process and inactivation of proteins.

that is general guidelines, and i say again i don't work on blood.

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Qiagen sells a nice RNA blood isolation kit. It worked for me. (mus tail bleeds and human blood)
Check their website for more info on yield, etc.

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The best kit I have used recently for isolating RNA from blood is the PAXGene kit from Qiagen. The blood is collected into or mixed with a special reagent (like RNAlater) and then you just follow the kit ... no RBC lysis or trying to get them WBC.

I have used 1-5 ml blood with good yield (enough to screen 8 genes by RT-PCR and have plenty left over) from both pig and human. Many have used for other animals.

Hope this helps :-0

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A few points to add:

1ml of whole blood will give you 1-5ug of total RNA, depending on the animal

PAXgene tubes can only be used with a fixed volume of 2.5ml of blood. PAXgene is a positively charged detergent which causes the RNA and DNA to precipitate out of solution. Nucleic acid is then pelleted by centrifugation and purified

Trizol BD is a differently formulated solution designed for liquid samples - you can use on the whole blood sample, eliminating loss by trying to separate WBCs

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Thanks gang. Appreciate it.
I was perusing the Qiagan catalogue earlier and thought that might be the way to go.
That PAX gene kit is spendy though.

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PAXgene is only really necessary if you can't do RNA isolation just after the blood is taken. The blood is drawn in the the vacuatainer, then has to be incubated overnight at 4C before you can process it

If you can go straight ahead with RNA isolation, just get the normal RNeasy kit (blood module) or IVGN micro-to-midi

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Were u able to extartx RNA from whole blood...?

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Hi, I need to do the same, qRT-PCR screening mouse RNA from tail bleeds, so far I'm looking for the best kit. Only one suitable for small volumes of blood is QIAamp RNA Blood Mini Kit, but it don't have anything for RNA stabilisation and we are looking for a low abundant transcript there.

PAXgene tubes offer that, but the're not designed for such small samples. Have anyone tried to use PAXgene (aliquoted to 1.5ml tubes perhaps) mixture for isolation of RNA from small blood volumes (let's say about 50ul)?

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