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Saturday, September 1, 2012

Cellular total RNA extraction from a ALL(Acute lymphocytic leukemia) patient's cells

There are two mainly way to extract RNA from cells.
    1. Use guanidinium thiocynate-phenol-chloroform to extract (traditional way)
    2. Silica column chromatography

Today, we choose traditional way to extract RNA from  a ALL(Acute lymphocytic leukemia) patient's cells. ALL is a form of leukemia, it may be caused by metabolic-genetic disorders,so we can know if a person would be a ALL patient by genomic tests.

First, we take 0.5ml TRIzol reagent(a mixture of guanidinium thiocyanate and phenol ) into an eppendorf with 0.17ml sample. Add 200uL chloroform into the eppendrof and mix it well then put the eppendrof on o ic bath for five minutes.

TRIzol reagent

In this reagent, I listed the using and properties of each chemical compound:

        Guanidinium thiocyanate:
         Guanidinium thiocyanate is a substance used as a general protein denaturant. It is also used to lyse cells and during lysing cell is to prevent activity of RNase enzymes by denaturing them.

        Phenol dissolves in water would be a weak acid. RNA has a lot of dipoles, so it is readily dissolve into an acidic and polarity phase.

         It is a kind of organic solvent. It provide an organic phase let DNA to dissolve in. It could also denature protein and protein would float on the face of chloroform. Otherwise, RNA would dissolves in water phase and the density of chloroform is higher then water, so we can suck out the upper phase(water) easily by a pipetment.

After five minutes, put the eppendrof in a refrigerated centrifug with 12000rpm, 4℃,ten minutes.

The white fuffy between phases is protein.
(I sucked out the upper phase so it looks no so much) 

Suck the water phase into a new ependrof, and add the same volume(as water phase) of isopropanol into the eppendrof. Then put the eppendrof on a ice bath for 45 minutes. In this process, RNA would precipitate.

Then put the eppendrof in a refrigerated centrifug with 12000rpm, 4℃,ten minutes.

 RNA is under the eppendrof

Pour out  all the isopropanol inside the eppendrof, and add 1ml 75% ethanol and then put the eppendrof in a refrigerated centrifug with 12000rpm, 4℃,ten minutes.

Pour out all the ethanol inside then put the eppendrof in a vacuumed centrifug to vapor out all ethanol.

A vacuumed centrifug is running

Add 20uL DECP water into the eppendrop to dissolve RNA.

Suck out 4uL RNA solution and mix it with 996uL DECP water , and measure OD260/280 nm

A UV-Visual spectrometer(Just need 1.0uL sample)

260/280 means the purity of RNA
260/230 means the containing of organic solvent

Finally, we got a 2617.8 ng/uL RNA sample from a ALL patient's cells.

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