RNA purification from tissue

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NB! pre-PCR operation


    • check, that you have:
    » liquid nitrogen
    » mortar and pestle
    » syringe and 19G needle
    • it is necessary to know the weight of the tissue

    [править] Homogenization

  1. select mortar:
    weight of the tissue [g]mortar diameter [cm]
    >116
    0.2-112.5
    <0.211

  2. NB! tissue disruption should be done unde fume hood[1]

  3. put mortar on foam plastic lid
  4. precool mortar and pestle with liquid nitrogen;
  5. freeze tissue in liquid nitrogen directly in mortar;
  6. grind in tiny powder;
  7. add liquid nitrogen[2] and desired volume of GTB: normally, ~20ml per 1g of tissue (see also table in Notes);
  8. grind in tiny powder;
  9. transfer powder into a tube;

  10. melt[3];
  11. homogenize and shear DNA[4] by 3-4 passing through ~19G needle;
  12. (optional, for tissues with high lipid content)
    • centrifuge 4krpm, room temperature, 10min
    • remove upper (lipid) phase

  13. [править] Acid phenol extraction

  14. ○○   two acidic phenol extraction:
    • add 1x V of acidic phenol and mix
    • incubate ~5 min. at room temperature
    • add 0.2x V of Ch/I[5], vortex for 1-3min.
    • centrifuge 4krpm, room temperature, 10min.
    • transfer the upper (aqueous) phase to a new tube
  15. chloroform extraction:
    • add 1x V of Ch/I[5], vortex for 1-3min.
    • centrifuge 4krpm, room temperature, 10min.
    • transfer the upper (aqueous) phase to a new tube

  16. [править] Precipitation

  17. add 1x V of Isopropanol, mix;
  18. incubate at -20°C 1h-overnight;
  19. centrifuge 4krpm, 4°C, 10min.;
  20. wash pellet with 70% EtOH;
  21. dilute in nuclease-free water[6];
  22. snap freeze in liquid nitrogen, store at
    • short-term: -20°C
    • long-term: -70°C




[править] Solutions

[править] GTB (Guanidine Thiocyanate Buffer)

ρ = 1.111
store at -20°C in 50ml aliquots

conc.stock50ml
Guanidine thiocyanate4M118.16g/M23.63g
Na citrate, pH 7.025mM1M1.25ml
N-Lauroylsarcosine Na0.5%10%2.5ml
H2O mQ27.8ml
β-MeEtOH*0.1M14.4M347µl
AcONa, 3M pH5.0*0.27M3M5ml

 * add β-MeEtOH and AcONa just before use


[править] Na citrate, Na3C6H5O7x2H2O

ρ = 1.17
store at 4°C

conc.stock50ml
Na3C6H5O7x2H2O1M294.1g/M14.71g
H2O mQ43.8ml




Acidic phenol


[править] Notes

  • for ~30g mouse:
OrganRNA
nameGTB [ml/g][7]weightcontent [µg/mg]per mouse
liver20~1.9g1120.9mg
spleen50180mg5-60.9mg
2 kidneys20450mg31.4mg
hart20180mg1180µg
lungs20200mg1.9380µg
tongue20120mg1120µg
2 prepuital glands2065mg2.7180µg
muscles15~1g0.8-1.3~1mg
2 testicules25250mg1.3320µg
2 seminal vesicules20120mg1.5180µg
mammary glands?
2 ovaries20?35mg2.270µg
uterus20?140mg3.7500µg
stomach200.3g1.80.54mg
colon200.53g3.72mg
intestine201.3g4.25.5mg
lymph nodes L1-L550-70120mg3.4400µg
thymus (young mice)50-70100mg2.1210µg
2 harderian glands30?20mg3.264µg
2 salivary glands[8]30150mg6.61mg
2 olfactory lobes (brain)[8]3030mg1.3-240µg
2 cerebral hemispheres (brain)[8]30230mg1.3-20.3mg
2 cerebellum[8]3060mg1.3-278µg
medulla[8]30150mg1.3-20.2mg
adrenal glands3035mg0.931µg


OrganRNA
blood1-5µg/ml
oocyte0.4ng
32-cells stage1.5ng
10 days embrio650µg
tails, ear~0.5µg/mg


  • for tissues with high lipid content it is worth to perform extraction with higher amount of chloroform. Or centrifuge lisate before phenol extraction.



  1. it is possible to catch a strong alergy if do it on the table
  2. be accurate, powder is volatile
  3. at 20-50°C
  4. DNA is sheared to ~50kbp
  5. 5,0 5,1 Chloroform/Isoamil alcohol = 24:1
  6. do not use DEPC-treated H2O
  7. GTB volume in the table is minimal. The more GTM, the better. For example, extraction of 1g liver in different volume of GTB
    • in 50ml GTB gives ~10mg RNA
    • in 5ml GTB — ~3.5mg RNA
  8. 8,0 8,1 8,2 8,3 8,4 remove lipids first


Личные инструменты


Инструменты




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