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TriPure Isolation Reagent Protocol

Product No. TRIPURE-RO

Protocol

Isolation of nucleic acids and proteins from plant sample material, e.g. leafs

Roche has not tested this experimental protocol that has reportedly worked successfully for at least Dandelion and Philodendron samples. In general, yields are better for plants having thin leaf walls and relatively lower for plants with leafs of high water content.

General Procedure:

1) Generally use between 100-300 mg leaf tissue per ml of TriPure Isolation Reagent.

2) Grind the leaf material in liquid nitrogen using mortar and pestle. This involves putting the leaf tissue into the mortar, adding a little of liquid nitrogen and crushing it with the pestle. Then add some more liquid nitrogen and crush some more. Do this at least one more time.

3a) TriPure Isolation Reagent can then be added directly to the mortar. Once the TriPure Isolation Reagent is added to the cold mortar, it will freeze immediately. Wait until the TriPure Isolation Reagent starts to thaw (this may take 5-15 min.) and becomes slushy. Use the pestle to crush the sample one final time.

3b) As an alternative to step 3a), the leaf powder can be added to a tube containing TriPure Isolation Reagent. This might be of advantage if large numbers of samples are being processed, because of the time to let the TriPure Isolation Reagent to thaw. Once the sample powder has been added to the TriPure Isolation Reagent reagent, final homogenization in a Dounce Tissue Grinder can be done.To extract DNA, do not use a Dounce Tissue Grinder, because of the likelihood of shearing the DNA. In general, for best results, perform step 3a as the final homogenization in the mortar.

4) After addition of chloroform, centrifuge the lysate (12,000 x g; 15 min) to separate the phases. Proceed with the isolation procedures as described in the product Package Insert.

Remarks:

I) If the leaf to be used has a coarse central stem, then it is not recommended to use a Dounce Tissue Grinder. The fibrous material of the central stem will make homogenization rather difficult.

II) Polytron® tissue homogenizer (or other sharp blade homogenizers) has not been tried, but will probably work well especially if the leafs have a coarse central stem.III) To isolate protein, for best results use less then the 100-300 mg leaf tissue per ml of TriPure Isolation Reagent (higher concentrations of leaf tissue might saturate the TriPure Isolation Reagent and result in lower yields of protein).

IV) Because of possible proteoglycan and polysaccharide contamination in RNA preparations, modify the RNA precipation step:- To the aqueous phase add 0.25 ml of isopropanol and 0.25 ml of high salt precipitation solution (0.8 M sodium citrate and 1.2 M NaCl) per ml of TriPure Isolation Reagent used in homogenization;- mix the solution;- centrifuge;- modification for RNA precipitation effectively precipitates RNA while maintaining proteoglycans and polysaccharides in solution.

Protocol for isolation RNA from fatty tissue and fiber structure using TriPure Isolation Reagent

For fatty tissues or fibrious tissues, such as muscle tissues, the procedure I for Extraction of RNA, DNA, and Protein Form Tissues or Cells can be followed, and especially the optional step 4 of Procedure 1 as described in the package insert of TriPure Isolation Reagent should be performed.

DNA contamination in isolated RNA using TriPure Isolation Reagent

Possible reasons for the contamination are:

  1. Insufficient TriPure Isolation Reagent used for sample homogenization: the recommendation would be to add a sufficient volume of TriPure Isolation Reagent
  2. The starting samples contained organic solvents (EtOH, DMSO), strong buffers, or had an alkaline pH: the recommendation would be to carefully remove the upper aqueous phase for subsequent RNA isolation, making sure to avoid the interphase/organic phase.

Materials

     

TRIPURE is a trademark of Roche

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