Isolation of RNA from tissue

/Isolation of RNA from tissue
Isolation of RNA from tissue 2018-02-23T12:23:41+00:00

Isolation of RNA from tissue

Isolation of RNA from tissue

Introduction

This protocol (CHOMCZYNSKI, 1993) allows the simultaneous recovery of RNA, DNA & protein from an aliquot of tissue or cells. This method involves lysis of cells with monophasic solution of guanidine isothiocynate and phenol. Addition of chloroform generates a second (organic) phase into which DNA & Proteins are extracted leaving RNA in aqueous supernatant. The RNA is precipitated from aqueous phase with isopropanol and can be further purified by chromatography on oligo dT- cellulose columns and/or used for northern blot hybridization, reverse transcription or RT-PCR. The yield of total RNA depends on the tissue or cell source, but it is generally 4-7 g/mg starting tissue or 5-10 g/ 10 6 cells. The DNA and Proteins can be isolated from the organic phase by sequential precipitation with ethanol and isopropanol respectively.

Requirements

Reagents

  1. TRI REAGENT (Sigma)
  2. Chloroform
  3. Ethanol
  4. DEPC treated water
  5. Isopropanol
  6. Ependorff tubes (1.5 ml & 2 ml)
  7. Ependorff tubes’ stand
  8. Micropipettes (1ml, 2-200l)
  9. Micro tips
  10. Polythene gloves
  11. Steel container (1l)
  12. Icebox
  13. Tissue

Preparation of Reagents

  1. Ethanol (75%): Diluted 75 ml of absolute ethanol to 100 ml with DEPC treated water. 
  2. Treatment of water with DEPC Added 0.1% DEPC (Sigma v/v) to 1 L Millipore water/triple distilled water. Bottle is sealed with Paraffin tape and covered with aluminum foil. Stir overnight on a magnetic stirrer at room temperature. The treated water was autoclaved for 30 minutes at 15psi.to remove the DEPC. 
  3. Treatment of microtips, ependorff tubes and PCR tubes with DEPC Added 0.1% DEPC solution to tip boxes (lower half), turn these upright down 3-4 times so that we can rinse the tips with DEPC water (seal tip boxes with parafilm), and fill ependorff tubes, PCR tubes with DEPC water. Put these tubes in a steel container (already baked for 5-6 hours at 180C), after filling with DEPC water. Put tip boxes and steel container on an orbital shaker for 7-8 hours. Autoclaved for 30 minutes at 15psi (wrap all the things with aluminium foil and paper before putting into autoclave).

Note: Don’t treat with DEPC more than one time and don’t autoclave for more than 2 times. Always wear polythene gloves while doing RNA work

Equipments

  1. Cooling centrifuge
  2. Vortex shaker
  3. Autoclave
  4. Pestle and mortar
  5. Plastic/glass Homogenizer
  6. Motor with regulator

Procedure

  1. Take 30-50 mg of tissue in liquid nitrogen (either preserved or taken from slaughterhouse). Crush to form powder with pestle and mortar with small volume of liquid nitrogen (be cautious while taking the sample from liquid nitrogen, use forceps). In case, if you are taking fresh tissue, dissect it wash it with saline and then lyse directly with TRI REAGENT.
  2. Add 1 ml of TRI REAGENTTM to powdered tissue sample, in two lots of 500l each. Homogenize it with polytron homogenizer.(you can use plastic homogenizer instead of polytron in case of fresh tissue)
  3. Centrifuge it 5 minutes at 12,400 rpm at 4C to remove any insoluble material (extra cellular membranes, polysaccharides and high molecular weight DNA).
  4. To supernatant add 0.2 ml of chloroform/ml of TRIreagent added.
  5. Cover tightly and shake vigorously for 15 seconds.
  6. Keep for 10-15 min. at room temperature.
  7. Centrifuge at 12,400 rpm for 15 min. at 4C.
  8. Transfer aqueous phase to a fresh tube.
  9. Added 500l of isopropanol to aqueous phase and mix well.
  10. Mix and allow the sample to stand for 10 min. at room temperature.
  11. Centrifuge at 12,400 rpm for 10 min. at 4C.
  12. Remove supernatant and wash the RNA pallet by adding 1 ml 75% ethanol and vortex to mix properly.
  13. Centrifuge at 9,800 rpm for 5 min. at 4C.
  14. Remove supernatant and dissolve pellet in 1 ml of 75% ethanol and store at -20C.
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