Do you know how to extract RNA ? - The Veterinary Site

Do you know how to extract RNA ?


RNA

(by MAAZ AHMED SIDDIQUI)
·         Stands for ribonucleic acid
·         Is a molecule essential in various biological roles in coding, decoding and expression of genes.

RNA STRUCTURE:

·         Is made up of molecules called nucleotides
·         Each nucleotide contains:
      -phosphate group
      -ribose sugar
      -nitrogenous base
·         Four types of bases are:

    -Adenine (A)                        -Uracil (U)
    -Guanine (G)                         -Cytosine (C)
·         Three major types of RNA are:
ü  Messenger RNA (mRNA) conveys genetic information that directs synthesis of specific proteins.
ü  Transfer RNA (tRNA) molecules deliver amino acids to the ribosome.
ü  Ribosomal RNA (rRNA) links amino acids together to form proteins.

PROTOCOL OF RNA EXTRACTION

RNA EXTRACTION:-


1.      Take 250ul of blood in an Eppendorf tube and add 250ul of DEPC water and 700-750ul of TRIzol.
2.      Shake it and put it for 5min at room temperature.
3.      After that add 200ul of chloroform.
4.      Shake for 10-15 seconds vigorously and place it at room temperature for 10min.
5.      Set the centrifuge on 40C at 13000rpm for 15min.
6.      Remove the samples from centrifuge.
7.      Extract the aqueous phase in which RNA is present carefully.
8.      Then add 500ul chilled isopropanol in the extracted RNA.
9.      Place it for 10min at room temperature.
10.  Centrifuge it again for 10min at 12000rpm at 40C.
11.  Discard the isopropanol, leaving 1/3rd of it in the eppendorph tube.
12.  Add 1ml of chilled 75% DEPC ethanol and mix well.
13.  Centrifuge then for 5min at 7500rpm at 4oC.
14.  Then discard the ethanol and dry the pellet for 10min only.
15.  After drying the pellet for 10min 25-30ul DEPC treated water is added and store it in -20oC in refrigerator.

 

What each reagent is for?


DEPC water:
·         Di ethyl pyrocarbonate 
·         Is used in the laboratory to inactivate RNase enzyme
·         Reduces the risk of RNA being degraded by RNases.
TRIzol:
·         A ready-to-use reagent used for RNA isolation from cells and tissues.
·         Simultaneously solubilize biological material and denature proteins. 
 Chloroform:
·         Causes phase separation 
·         Where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase.
Chilled isopropanol:
·         Causes precipitation
Ethanol:
·         To wash away the salts 


Do you know how to extract RNA ? Do you know how to extract RNA ? Reviewed by Maaz ahmed siddiqui on 10 May Rating: 5

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