How many neuts for a cDNA prep?

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Jason King
Jason King's picture
How many neuts for a cDNA prep?

Hi,

I routinely use a M&N Nucleospin RNA II kit to make total RNA (which I then use for cDNA and qPCR).

Just recently I did a flow sort of neutrophils to get a million neuts (free of eosinophils) from which I had hoped to make RNA. Amazingly, the spec' suggested that absolutely no RNA had be purified from these million cells.

Neuts are known to be transcriptionally "quiet", but I thought I'd get something out of it.

Does anyone know how many neuts to start with?

Omai
Omai's picture
Hi parvoman,

Hi parvoman,

I don't have any personal experience, but i found this paper that isolates RNA from neutrophils for qPCR.

Here is the website:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=551605

Hope this helps.

It looks like they use a different RNA isolation kit. RNeasy Mini Kit (Qiagen). Maybe this would help.

Cell count wise, they just say "20 ml of peripheral blood was taken into heparinized tubes. Neutrophil isolation was performed by a Dextran-Ficoll sedimentation and centrifugation method"

Unfortunately they don't give a total cell number.

Omai

Jason King
Jason King's picture
Thank Omai,

Thank Omai,

20mL of blood would yield far more than 1 million neutrophils.

I've just repeated the RNA prep and got a measurable concentration so this should be enough to get cDNA using Stratagene's cDNA kit.