RNA Extraction

Total RNA Extraction with Spectrum™ Plant Total RNA Kit (STRN50)

Materials:

Reagents Provided	Catalog No.	50 Preps
Lysis Solution	L8167	50 ml
2-Mercaptoethanol	M3148	0.9 ml
Binding Solution	L8042	50 ml
Wash Solution 1	W1141	50 ml
Wash Solution 2 Concentrate*	W3261	15 ml
Elution Solution	E8024	10 ml
Filtration Columns	C6866	50 each
Binding Columns	C6991	50 each
Collection Tubes, 2 ml	T5449	4 x 50 each

Reagents and Equipment Required But Not Provided
Mortar and Pestle	Microcentrifuge
Liquid Nitrogen	Heat block
Dry Ice	100% Ethanol
2 ml microcentrifuge tubes for weighing tissue	DNase I

Preparation:

1. Clean the area with RNA Zap
2. *Dilute Wash Solution 2

Add 60 ml of 100% Ethanol to the 50 prep kit size wash solution 2 bottle and store the diluted solution, tightly capped. 

3. Grind Plant Tissue (Previously Collected, Frozen, and Stores at -80 C)

Grind the tissue in a mortar and pestle rested on dry ice and keep the plant material frozen at all times.

4. Weigh Tissue Sample

Weigh 100 mg (90-110 mg) of the tissue powder in a 2-ml microcentrifuge tube pre-chilled on dry ice on liquid nitrogen. Keep the sample on dry ice until the lysis solution is added. 

5. Prepare Lysis Solution/2-ME Mixture

In a clean conical tube combine 10 ul of 2-ME for every 1 ml of Lysis solution and mix briefly. Each RNA preparation requires 0.5 ml (500 ul) of the mixture. Do not store for more than 1 day. 

Sample size	2-ME (ul)	Lysis Solution (ml)
1	10	1
5	50	5
10	100	10
20	200	20
50	500	50

6. Assemble column and Collection Tube

Insert a filtration column (blue retainer ring) into a 2-ml collection tube and close the lid (used for Bind RNA). Insert a binding column (red retainer ring) into a 2-ml collection tube and close the lid (used for Filter Lysate).

Procedure:

Note: All centrifuge steps are performed at room temperature and t max speed (14,000-16,000 x g) in a standard centrifuge 

1. Lyse Tissue Sample

Pipette 500 ul of Lysis Solution/2-ME Mixture to 100 mg of tissue powder. 

Vortex immediately for 30 seconds. 

Incubate the sample for 3-5 minutes at 56 C. 

2. Pellet Cellular Debris

Centrifuge at max speed for 3 minutes. Mark the tissue orientation.

3. Filter Lysate

Pipette the lysate supernatant into a filtration column (blue retainer). Close the cap and centrifuge for 1 minute. Save the clarified flow-through lysate. Re-centrifuge for 3-5 minutes if the liquid has not passed through the filter. 

4. Bind RNA to Column

Pipette 500 ul of Binding Solution into the clarified lysate and mix immediately and thoroughly by pipetting at least 5 times. 

Pipette 700 ul of the mixture into a BInding Column (red retainer), close, and centrifuge for 1 minute. Decant the flow-through and tap the collection tube (upside down) on a KimWipe to drain. Return the column to the collection tube and pipette the remaining mixture to the column and repeat centrifuge and decant steps. 

5. On-Column DNase Digestion

Pipette 300 ul of Wash Solution I into the Binding Column, close cap, and centrifuge for 1 minute. Decant the flow-through, tap the collection tube on a KimWipe, and return the column to the collection tube. 

For each digestion, combine 10 ul of DNase I with 70 ul of DNase digestion buffer and mix gently by pipetting. 

Pipette 80 ul of the mixture directly onto the center of the filter inside the Binding column, close the cap, and incubate at room temperature for 15 minutes.

6. Column Wash 1-3

Pipette 500 ul Wash Solution I into the Binding Column, close the cap and centrifuge for 1 minute. Decant the flow-trough, tap the collection tube on a KimWipe and return the column to the collection tube. 

Pipette 500 ul of the diluted Wash Solution II into the Binding Column, close the cap and centrifuge for 30 seconds. Decant the flow-trough, tap the collection tube on a KimWipe and return the column to the collection tube. 

Pipette 500 ul of the diluted Wash Solution II into the Binding Column, close the cap and centrifuge for 30 seconds. Decant the flow-trough, tap the collection tube on a KimWipe and return the column to the collection tube. 

7. Dry Column

Centrifuge the column for 1 minute.

8. First Elution

Transfer the column to a new, clean 2 ml Collection tube. Pipette 50 ul of Elution Solution directly onto the center of the binding matrix inside the column. Close the cap and let the tube sit for 1 minute. Centrifuge for 1 minute. Store the flow-through at -20 C (short term) or -70 C (long term). Repeat this step with a new, clean 2 ml Collection tube if expected RNA yield is >20 mg.

Analysis of RNA

1. Use NanoDrop to obtain concentration and quality of total RNA.
2. Gel electrophoresis.

Total RNA Extraction with TRIzol

Item

Equipment

Water bath or heat block at 55-60°C

Reagents

Isopropanol

Ethanol, 75%

RNase-free water of 0.5% SDS

[Optional] RNase-free glycogen

1. Lyse samples and separate phases

• Add 1 mL of TRIzol Reagent per 50–100 mg of tissue to the sample and homogenize using a homogenizer.
• Centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.
• Incubate for 5 minutes to allow complete dissociation of the nucleoproteins complex.
• Add 0.2 mL of chloroform per 1 mL of TRIzol Reagent used for lysis, securely cap the tube, then thoroughly mix by shaking.
• Incubate for 2–3 minutes.
• Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.
• Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.

2. Precipitate the RNA

• Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzol Reagent used for lysis. 
• Incubate for 10 minutes at 4°C.
• Centrifuge for 10 minutes at 12,000 × g at 4°C.
  • Total RNA precipitate forms a white gel-like pellet at the bottom of the tube. 
• Discard the supernatant with a micropipette.

3. Wash the RNA

• Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol Reagent used for lysis.
  • Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C.
• Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.
• Discard the supernatant with a micropipette.
• Vacuum or air dry the RNA pellet for 5–10 minutes.

4. Solubilize the RNA

• Resuspend the pellet in 20–50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down.
• Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.