Mrna Isolation Using Oligotex Sin-Column

Mrna Isolation Using Oligotex Sin-Column

Total RNA Isolation from Rice Tissue

Materials and equipment necessary for this protocol:

TRIzol (Invitrogen)

RNeasy Kit, Mini (Qiagen)

Contained in this kit: columns, collection tubes, RW1 Buffer, RPE Buffer, RNase-Free water

RNase-Free DNaseI

Chloroform, 70% ethanol, 10 mM Tris-Cl pH 7.5 (RNase-Free)

50 ml conical polypropylene tubes, 17 mm snap-cap polypropylene tubes, Kimwipes

Liquid Nitrogen

Note: Using this protocol we typically obtain 1mg of total RNA from 2-3 grams of leaf tissue. Typical mRNA yields are 5-10ug. Labeling reactions require 1ug of mRNA for each sample and 2ug of mRNA if doing dye swaps. This protocol can be scaled up or down as necessary.

Protocol:

  1. Remove rice tissue and place immediately in liquid nitrogen. Tissue can be stored frozen at –80oC until ready to use.
  2. Grind 2-3 grams frozen rice tissue using a mortar and pestle. Add additional liquid nitrogen and grind to a fine powder. Repeat 3 times to obtain a very fine powder. Additional grinding steps greatly increase RNA yield. (Do not let tissue thaw at anytime during this process).
  3. Add tissue directly to 40ml falcon tube containing 20 – 30 mL Trizol reagent.

(Invitrogen recommends 10ml of Trizol per 1g of tissue)

  1. Allow samples to sit for at least 5 min before proceeding.

(The Samples in this step can be stored at –80oC for six months. Important: Samples before adding chloroform)

  1. Add 2mL of chloroform to each tube. Mix by vigorous inversion for 15 to 30 seconds. Let sit for 2 – 3 min. (2mL of chloroform / 10 mL of Trizol)
  2. Centrifuge at 12,000g for 10 min. Transfer aqueous phase (top) to a fresh centrifuge tube. Add 5mL of isopropanol to the tube and mix. Spin at 12,000g for 10min. Remove supernatant and wash pellet with 75% ethanol. Mix and centrifuge at 7,500g for 5 min. Resuspend RNA in 425uL Nuclease Free water.

(If the tissues are from old plants, it is better to do chloroform extraction one time before isopropanol precipitation. Add same volume of chloroform to aqueous phase obtained)

  1. Add 50uL DNaseI buffer (10x) and 25uL DNaseI. Incubate at room temperature for 30min. (No longer than 30 min)
  2. Add 2mL buffer RLT and 1.4mL 100% ethanol.

(Be sure that BME is added to RLT buffer 100uL BME/ 50 mL RLT)

  1. Add sample to an RNEasy midi column (columns bind maximum 1mg of RNA). Spin column at 3,000-5,000g for 5 min to flush column (discard flow-through between spins). Re-use the collection tube.
  2. Add 2.5mL Buffer RPE to each spin column. Centrifuge at 3,000-5,000g for 2 min. Discard the wash. Re-use the collection tube. Repeat wash with 2.5mL RPE and spin for 5min at 3,000-5,000g to dry column.
  3. Add 250 µl RNase-Free water to each column. Let stand for 1 min and spin at 3,000-5,000g for 5 min. DO NOT DISCARD ELUANT.

(Before this step, turn on the water bath and adjust temperature to 65 oC. Heat water up and add hot water to each column to maximize the efficiency of the elution and stand it for 10 min. in water bath.)

  1. Add another 250 µl RNase-Free water to each column. Let stand for 1 min and spin at 3,000-5,000g for 5 min.
  2. Final elution volume is 500 µl. Discard column. DO NOT DISCARD ELUANT. Leave on ice.

(500 µl of RNase-Free water can be used for each time instead of 250 µl if RNA is enough because 500 µl of water will be added in the first step of mRNA purification)

  1. Quantify samples by obtaining absorbance at 260 and 280 nm. Dilute 1 µl of total RNA sample 1:100 using 10 mM Tris-Cl pH 7.5 (1 µl RNA + 99 µl buffer) and mix well by pipetting. Obtain absorbance readings using a clean microcuvette using the diluted sample. Discard diluted samples once absorbance has been obtained. Use the accompanying RNA quantitation template to calculate RNA yields and purity.
  2. Depending on yield, prepare 1 – 2 µl RNA sample for gel electrophoresis by combining with appropriate loading buffer.
  3. Total RNA samples should be stored at -80ºC.
  4. Procede to Oligotex mRNA purification protocol.
Top View