50 prep

Introduction and Overview

E.Z.N.A.® Fungal RNA Mini Kit provides a rapid and reliable method for isolation of total RNA from a wide variety of fungal samples. This kit does not require the use of cumbersome or expensive shredding/homogenizing accessories as an attempt to shear viscous fungal lysates. Rather, this method involves a simple and rapid precipitation step for removal of much of the polysachrides and phenolic compounds commonly found in fungal tissues. In combination with HiBind® RNA Mini Column, this method permits purification of high-quality RNA from as much as 200 mg tissue. The system is efficient enough to allow total RNA from as little as 10 mg tissue or 100 cells. Typical yields are shown below in Table 1. The procedure involves no organic extractions, thus reducing plastic waste and hands-on time. E.Z.N.A.® Fungal RNA Mini Kits are ideal for processing multiple fungal samples in parallel in 1 hour. Purified RNA has A260/A280 ratios of 1.8-2.0 and is suitable for the following applications:

• RT-PCR

• Northern analysis

• Differential display

• Poly A+ RNA selection

Table 1. Yields obtained with E.Z.N.A.® Fungal RNA Mini Kit

Acremonium chrysogenum	50 μg
Fusarium avenaceum	37 μg
Mushrooms	43 μg

Binding Capacity:

Each HiBind® RNA Mini Column can bind approximately 100 μg RNA. Using greater than 250 mg fungal tissue in many cases will not dramatically improve yields and sometimes has adverse effects

New In this Edition

November 2018:

• DEPC Water has been replaced with Nuclease-free Water. DEPC Water is no longer provided in this kit.

• PR032 (DEPC Water, 100 mL) has been discontinued and is no longer available to purchase.

May 2014:

• NTL Lysis Buffer has replaced RFL Buffer to enhance lysis and improve yields

Quantification of RNA

Quantification and Storage of RNA

To determine the concentration and purity of RNA, measure absorbance at 260 nm and 280 nm with a spectrophotometer. One OD unit measured at 260 nm corresponds to 40 μg/mL RNA. Nuclease-free Water is slightly acidic and can lower A260/A280 ratios. Use TE buffer to dilute RNA prior to spectrophotometric analysis. The A260/A280 ratio of pure nucleic acids is 2.0, while an A260/A280 ratio of 0.6 denotes pure protein. A ratio of 1.8-2.0 corresponds to 90%-100% pure nucleic acid. Phenol has a maximum absorbance at 270 nm and can interfere with spectrophotometric analysis of DNA or RNA. Store RNA samples at -70°C in water. Under these conditions, RNA is stable for more than a year.

Integrity of RNA

It is highly recommended that RNA quality be determined prior to beginning all downstream applications. The quality of RNA can be best assessed by denaturing agarose gel electrophoresis with ethidium bromide staining. The ribosomal RNA bands should appear as sharp, clear bands on the gel. The 28S band should appear to be double that of the 18S RNA band (23S and 16S if using bacteria). If the ribosomal RNA bands in any given lane are not sharp and appear to be smeared towards the smaller sized RNA, it is very likely that the RNA undergone degradation during the isolation, handling, or storage procedure. Although RNA molecules less than 200 bases in length do not efficiently bind to the HiBind® matrix, a third RNA band, the tRNA band, may be visible when a large number of cells are used.

Product	R6840-00	R6840-01
Purifications	5	50
HiBind® RNA Mini Columns	5	50
Homogenizer Mini Columns	5	50
2 mL Collection Tubes	15	150
NTL Lysis Buffer	5 mL	40 mL
SP Buffer	2 mL	10 mL
RB Buffer*	5 mL	30 mL
RNA Wash Buffer I	5 mL	30 mL
RNA Wash Buffer II	5 mL	12 mL
Nuclease-free Water	2 mL	30 mL
User Manual

* RB Buffer and NTL Lysis Buffer contain a chaotropic salt. Use gloves and protective eyewear when handling these solutions.

Storage and Stability

All of the E.Z.N.A.® Fungal RNA Mini Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature. During shipment or storage in cool ambient conditions, precipitates may form in RB Buffer. Dissolve such deposits by warming the solution at 37°C and gently shaking.

Preparing Reagents

• Dilute RNA Wash Buffer II with 100% ethanol as follows and store at room temperature.

Kit	100% Ethanol to be Added
R6840-00	20
R6840-01	48

• Add 20 µL 2-mercaptoethanol (β-mercaptoethanol) per 1 mL RB Buffer and/or NTL Lysis Buffer depending on the protocol. This mixture can be stored for 4 weeks at room temperature.

Important Notes

Please take a few minutes to read this booklet in its entirety to become familiar with the procedures. Prepare all materials required before starting to minimize RNA degradation.

• Whenever working with RNA, always wear gloves to minimize RNase contamination. Use only clean RNase-free disposable plastic pipette tips when using the supplied reagents.

• Equilibrate samples and reagents to room temperature before beginning this protocol. All steps should be carried out at room temperature unless otherwise noted. Work quickly, but carefully.

• Prepare all materials required before starting the procedure to minimize RNA degradation.

• Carefully apply the sample or solution to the center of the HiBind® RNA Mini Columns. Avoid touching the membrane with pipet tips.

• 2-mercaptoethanol is key in denaturing RNases and can be added to an aliquot of RB Buffer or NTL Lysis Buffer before use. Add 20 μL 2-mercaptoethanol per 1 mL RB Buffer or NTL Lysis Buffer. This mixture can be stored for 4 weeks at room temperature.

E.Z.N.A.® Fungal RNA Mini Kit Protocol

E.Z.N.A.® Fungal RNA Mini Kit Protocol - Standard Protocol

This protocol is suitable for most fresh or frozen tissue samples allowing for efficient recovery of RNA. However, due to the tremendous variation in water and polysaccharide content of fungal samples, sample size should be limited to ≤100 mg. The method isolates sufficient RNA for a few tracks on a standard Northern assay. Collect tissue in a 1.5 mL or 2.0 mL microcentrifuge tube and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube. Grind the tissue using disposable homogenization pestles (available from Omega Bio-tek, Cat# SSI-1015-39) or equivalent. Alternatively, one can allow liquid nitrogen to evaporate and then store samples at -70°C for later use. Do not allow the samples to thaw. Use disposable pestles only once. Alternatively, a small clean mortar and pestle can be used. The above methods for disrupting fungal tissue cannot be replaced with mechanical homogenizers.

Materials and Equipment to be Supplied by User:

• Microcentrifuge capable of 13,000 x g

• Nuclease-free 1.5 mL or 2 mL microcentrifuge tubes

• Incubator, heat block, or water bath capable of 65°C

• 2-mercaptoethanol

• 100% ethanol

• Liquid nitrogen for freezing/disrupting samples

• Optional: DNase I Digestion Set (E1091)

Before Starting:

• Prepare RNA Wash Buffer II and RB Buffer according to the “Preparing Reagents” section on Page 5

• Heat Nuclease-free Water to 65°C

Notes: Use extreme caution when handling liquid nitrogen. All centrifugation steps must be carried out at room temperature.

1. Transfer up to 100 mg frozen ground fungal tissue to a 1.5 mL or 2 mL microcentrifuge tube (not provided).

Note: We recommend starting with 50 mg of tissue at first. If results obtained are satisfactory then increase the amount of starting material.

2. Immediately add 500 μL RB Buffer. Samples should not be allowed to thaw before RB Buffer is added.

Note: RB Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 5 for instructions.

3. Vortex vigorously to make sure that all of the clumps have dispersed. RNA cannot be effectively extracted from a clumped sample.

4. Insert a Homogenizer Mini Column into a 2 mL Collection Tube.

5. Transfer the lysate to the Homogenizer Mini Column.

6. Centrifuge at 13,000 x g for 5 minutes at room temperature.

7. Carefully transfer the supernatant of the filtrate to a new 1.5 mL or 2 mL microcentrifuge tube. Do not disturb the pellet or transfer any debris.

Tip: In most cases 450 μL supernatant can easily be removed. This will require 225 μL 100% ethanol in the next step. Depending on the sample, the volume of supernatant may vary. After transferring to a new tube, measure the volume and add the correct amount of ethanol in the next step.

8. Add 0.5 volumes 100% ethanol. Vortex to mix thoroughly.

9. Insert a HiBind® RNA Mini Column into a 2 mL Collection Tube.

10. Transfer the entire sample from Step 8, including any precipitates that may have formed, to the HiBind® RNA Mini column.

11. Centrifuge at 10,000 x g for 30 seconds at room temperature.

12. Discard the filtrate and reuse the collection tube

Optional: This the starting point of the optional on-membrane DNase I Digestion Protocol. Since the HiBind® matrix of the RNA Mini Column eliminates most DNA, DNase I digestion is not necessary for most downstream applications. However, certain sensitive RNA applications may require further DNA removal. If an additional RNA removal step is required, please continue to the DNase I Digestion Protocol found on Page 15. (See DNase I Digestion Set, (E1091) for more information). If DNase I digestion is not required, proceed to Step 13.

13. Add 500 μL RNA Wash Buffer I.

14. Centrifuge at 10,000 x g for 30 seconds.

15. Discard the filtrate and the collection tube.

16. Transfer the HiBind® RNA Mini Column into a new 2 mL Collection Tube.

17. Add 500 μL RNA Wash Buffer II.

18. Centrifuge at 10,000 x g for 30 seconds.

Note: RNA Wash Buffer II must be diluted with 100% ethanol before use. Please see Page 5 for instructions.

19. Discard the filtrate and reuse the Collection Tube.

20. Repeat Steps 17-19 for a second RNA Wash Buffer II wash step.

21. Centrifuge at maximum speed for 2 minutes to completely dry the HiBind® RNA Mini Column.

Note: It is important to dry the HiBind® RNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.

22. Transfer the HiBind® RNA Mini Column to a clean 1.5 mL or 2 mL microcentrifuge tube (not provided).

23. Add 50-100 μL Nuclease-free Water.

Note: Make sure to add water directly onto the HiBind® RNA Mini Column matrix.

24. Centrifuge at top speed for 1 minute and store eluted RNA at -70°C.

Note: A second elution into the same tube may be necessary if the expected yield of RNA is >50 μg. Alternatively, RNA may be eluted with a greater volume of Nuclease-free Water. While an additional elution step will increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution.

 

E.Z.N.A.® Fungal RNA Mini Kit Protocol - Difficult Sample Types

Certain fungal samples are very difficult for RNA isolation because of the amount of material and type of secondary metabolites. This method involves a simple and rapid precipitation step for removal of much of the polysaccharides and phenolic compounds commonly found in fungal tissues. Use this protocol when standard protocol did not yield RNA or provided a lower yield.

Materials and Equipment to be Supplied by User:

• Microcentrifuge capable of 10,000 x g

• Nuclease-free 1.5 mL or 2 mL microcentrifuge tubes

• Incubator, heat block, or water bath capable of 65°C

• 2-mercaptoethanol

• 100% ethanol

• 70% ethanol

• Isopropanol

• Liquid nitrogen for freezing/disrupting samples

• Optional: DNase I Digestion Set (E1091)

Before Starting:

• Prepare RNA Wash Buffer II and NTL Lysis Buffer according to the “Preparing Reagents” section on Page 5

• Heat Nuclease-free Water to 65°C

Notes: Use extreme caution when handling liquid nitrogen. All centrifugation steps must be carried out at room temperature.

1. Transfer up to 100 mg frozen ground fungal tissue to a 1.5 mL or 2 mL microcentrifuge tube.

Note: We recommend starting with 50 mg of tissue at first. If results obtained are satisfactory then increase the amount of starting material.

2. Immediately add 600 μL NTL Lysis Buffer. Samples should not be allowed to thaw before NTL Lysis Buffer is added.

Note: NTL Lysis Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 5 for instructions.

3. Vortex vigorously to make sure that all of the clumps have dispersed. RNA cannot be effectively extracted from a clumped sample.

4. Add 140 μL SP Buffer. Vortex to mix thoroughly.

5. Centrifuge at 10,000 x g for 10 minutes at room temperature.

6. Carefully transfer the supernatant of the filtrate to a new 1.5 mL or 2 mL microcentrifuge tube. Do not disturb the pellet or transfer any debris.

Tip: In most cases 600 μL supernatant can easily be removed. This will require 600 μL isopropanol in the next step. Depending on the sample, the volume of supernatant may vary. After transferring to a new tube, measure the volume and add the correct amount of isopropanol in the next step.

7. Add 1 volume isopropanol. Vortex to mix thoroughly.

8. Immediately centrifuge at 10,000 x g for 2 minutes at room temperature. A longer centrifugation does not improve yields.

9. Carefully aspirate and discard the supernatant. Do not disturb the RNA pellet.

10. Invert the microcentrifuge tube on a paper towel for 1 minute to allow residual liquid to drain. Drying the pellet is not necessary.

11. Add 100 μL RB Buffer or Nuclease-free Water heated to 65°C. Vortex at maximum speed to resuspend the pellet. A brief incubation at 65°C may be necessary.

Note: RB Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 5 for instructions.

Important: RB Buffer is recommended to dissolve the RNA pellet at this step, particularly if degradation has been found after elution. RB Buffer contains a strong RNase inhibitor. If it is difficult to dissolve the RNA pellet with RB Buffer, use Nucleasefree Water instead.

12. This step varies depending on the buffer used to resuspend the RNA pellet in Step 11.

A. For RB Buffer:

1. Add 250 μL RB Buffer.

2. Add 350 μL 70% ethanol.

3. Vortex to mix thoroughly.

4. Proceed to Step 13.

B. For Nuclease-free Water:

1. Add 350 μL RB Buffer.

2. Add 250 μL 100% ethanol.

3. Vortex to mix thoroughly.

4. Proceed to Step 13.

Note: RB Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 5 for instructions.

13. Insert a HiBind® RNA Mini Column into a 2 mL Collection Tube.

14. Transfer the entire sample from Step 12, including any precipitates that may have formed, to the HiBind® RNA Mini column.

15. Centrifuge at 10,000 x g for 30 seconds at room temperature.

16. Discard the filtrate and reuse the collection tube.

Optional: This the starting point of the optional on-membrane DNase I Digestion Protocol. Since the HiBind® matrix of the RNA Mini Column eliminates most DNA, DNase I digestion is not necessary for most downstream applications. However, certain sensitive RNA applications may require further DNA removal. If an additional RNA removal step is required, please continue to the DNase I Digestion Protocol found on Page 15. (See DNase I Digestion Set, (E1091) for more information). If DNase I digestion is not required, proceed to Step 17.

17. Add 500 μL RNA Wash Buffer I.

18. Centrifuge at 10,000 x g for 30 seconds

19. Discard the filtrate and the collection tube.

20. Transfer the HiBind® RNA Mini Column into a new 2 mL Collection Tube.

21. Add 500 μL RNA Wash Buffer II.

22. Centrifuge at 10,000 x g for 30 seconds.

Note: RNA Wash Buffer II must be diluted with 100% ethanol before use. Please see Page 5 for instructions.

23. Discard the filtrate and reuse the Collection Tube.

24. Repeat Steps 21-23 for a second RNA Wash Buffer II wash step.

25. Centrifuge at maximum speed for 2 minutes to completely dry the HiBind® RNA Mini Column.

Note: It is important to dry the HiBind® RNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.

26. Transfer the HiBind® RNA Mini Column to a clean 1.5 mL microcentrifuge tube (not provided).

27. Add 50-100 μL Nuclease-free Water.

Note: Make sure to add water directly onto the HiBind® RNA Mini Column matrix.

28. Centrifuge at top speed for 1 minute and store eluted RNA at -70°C.

Note: A second elution into the same tube may be necessary if the expected yield of RNA is >50 μg. Alternatively, RNA may be eluted with a greater volume of Nuclease-free Water. While an additional elution step will increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution.

 

E.Z.N.A.® Fungal RNA Mini Kit - DNase I Digestion Protocol

Since the HiBind® matrix of the RNA Mini Column eliminates most DNA, DNase I digestion is not necessary for most downstream applications. However, certain sensitive RNA applications may require further DNA removal. (See DNase I Digestion Set, Cat# E1091 for further information).

After completing Steps 1-12 of the Standard Protocol (Pages 7-8) or Steps 1-16 of the Difficult Sample Types Protocol (Pages 11-13), proceed with the following protocol.

User Supplied Material:

• DNase I Digestion Set (E1091)

1. For each HiBind® RNA Mini Column, prepare the DNase I stock solution as follows:

Buffer	Volume per Prep
E.Z.N.A.® DNase I Digestion Buffer	73.5 μL
RNase-free DNase I (20 Kunitz/µL)	1.5 μL
Total Volume	75 μL

Important Notes:

• DNase I is very sensitive and prone to physical denaturing. Do not vortex the DNase I mixture. Mix gently by inverting the tube.

• Freshly prepare DNase I stock solution right before RNA isolation.

• Standard DNase buffers are not compatible with on-membrane DNase I digestion. The use of other buffers may affect the binding of RNA to the HiBind® matrix and may reduce RNA yields and purity.

• All steps must be carried out at room temperature. Work quickly, but carefully.

2. Insert the HiBind® RNA Mini Column containing the sample into a 2 mL Collection Tube.

3. Add 250 µL RNA Wash Buffer I to the HiBind® RNA Mini Column.

4. Centrifuge at 10,000 x g for 1 minute.

5. Discard the filtrate and reuse the Collection Tube.

6. Add 75 μL DNase I digestion mixture directly onto the surface of the membrane of the HiBind® RNA Mini Column.

Note: Pipet the DNase I directly onto the membrane. DNA digestion will not be complete if some of the mixture is retained on the wall of the HiBind® RNA Mini Column.

7. Let sit at room temperature for 15 minutes.

8. Add 250 μL RNA Wash Buffer I to the HiBind® RNA Mini Column.

9. Let sit at room temperature for 2 minutes.

10. Centrifuge at 10,000 x g for 1 minute.

11. Discard the filtrate and reuse the Collection Tube.

12. Add 500 μL RNA Wash Buffer II.

Note: RNA Wash Buffer II must be diluted with 100% ethanol before use. Please see Page 5 for instructions.

13. Centrifuge at 10,000 x g for 1 minute.

14. Discard the filtrate and reuse the Collection Tube.

15. Repeat Steps 12-14 for a second RNA Wash Buffer II wash step.

16. Centrifuge at maximum speed for 2 minutes to completely dry the HiBind® RNA Mini Column matrix.

Note: It is important to dry the HiBind® RNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.

17. Place the column in a clean 1.5 mL or 2 mL microcentrifuge tube (not supplied).

18. Add 50-100 μL Nuclease-free Water.

Note: Make sure to add water directly onto the HiBind® RNA Mini Column matrix.

19. Let sit at room temperature for 1 minute.

20. Centrifuge at maximum speed for 2 minutes and store eluted RNA at -70°C.

Note: A second elution into the same tube may be necessary if the expected yield of RNA is >50 μg. Alternatively, RNA may be eluted with a greater volume of Nuclease-free Water. While an additional elution step will increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution.

Troubleshooting Guide

Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact the technical support staff, toll free, at 1-800-832-8896.

Problem	Cause	Solution
Little or no RNA eluted	RNA remains on the column	• Repeat elution. • Heat Nuclease-free Water to 65°C prior to elution. • Incubate column for 10 minutes prior to centrifugation.
	Column is overloaded	Reduce the amount of starting material.
Problem	Cause	Solution
Clogged column	Incomplete disruption or lysis of fungal tissue	• Completely grind sample in liquid nitrogen. • Increase centrifugation time. • Reduce amount of starting material
Precipitated RNA will not dissolve	High nucleic acid and polysaccharide content	• Reduce amount of starting material. Start with 50-100 mg. • To avoid RNA degradation, use RB Buffer to dissolve the RNA pellet.
Problem	Cause	Solution
Degraded RNA	Source	• Freeze starting material quickly in liquid nitrogen and store at -70°C without thawing. • Follow protocol closely and work quickly. • Make sure that 2-mercaptoethanol is added to RB and/or NTL Lysis Buffer.
	RNase contamination	• Ensure not to introduce RNases during the procedure. • Check buffers for RNase contamination
Problem	Cause	Solution
Problem in downstream applications	Salt carryover during elution	• Ensure RNA Wash Buffer II has been diluted with 100% ethanol. • Diluted RNA Wash Buffer II must be stored at room temperature. • Repeat wash step with RNA Wash Buffer II
DNA contamination	Co-purification of DNA	Follow the DNase I Digestion Protocol on Page 15.
Low Abs ratios	RNA diluted in acidic buffer or water	Nuclease-free Water is slightly acidic and can lower A260/A280 ratios. Use TE buffer to dilute RNA prior to spectrophotometric analysis.

 

 

 

 

 

 

 

August 2019:

• DEPC Water has been replaced with Nuclease-free Water. DEPC Water is no longer provided in this kit.

• PR032 (DEPC Water, 100 mL) has been discontinued and is no longer available to purchase.

August 2014:

• Homogenizer Columns are now called Homogenizer Mini Columns. This is a name change only. No change has been made to the column itself.

Binding Capacity

• Each HiBind® RNA Mini Column can bind approximately 100 µg RNA. Using greater than 100 mg plant tissue is not recommended.

 

Kit Contents

Plant RNA Mini Kit	R6827-00	R6827-01	R6827-02
Preparations	5	50	200
HiBind® RNA Mini Columns	5	50	200
Homogenizer Mini Columns	5	50	200
2 mL Collection Tubes	15	150	600
NTL Lysis Buffer	5 mL	40 mL	130 mL
SP Buffer	2 mL	10 mL	40 mL
RB Buffer	5 mL	30 mL	110 mL
RNA Wash Buffer I	5 mL	50 mL	200 mL
RNA Wash Buffer II	5 mL	25 mL	50 mL
Nuclease-free Water	2 mL	30 mL	60 mL
User Manual

Storage and Stability

All of the E.Z.N.A.® Plant RNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature. During shipment or storage in cool ambient conditions, precipitates may form in RB Buffer. Dissolve such deposits by warming the solution at 37°C and gently shaking.

Important Notes

Please take a few minutes to read this booklet in its entirety to become familiar with the procedures. Prepare all materials required before starting to minimize RNA degradation.

• Whenever working with RNA, always wear gloves to minimize RNase contamination. Use only clean RNase-free disposable plastic pipette tips when using the supplied reagents.

• Equilibrate samples and reagents to room temperature before beginning this protocol. All steps should be carried out at room temperature unless otherwise noted. Work quickly, but carefully.

• Prepare all materials required before starting the procedure to minimize RNA degradation.

• Carefully apply the sample or solution to the center of the HiBind® RNA Mini Columns. Avoid touching the membrane with pipet tips.

• 2-mercaptoethanol is key in denaturing RNases and can be added to an aliquot of RB Buffer before use. Add 20 μL 2-mercaptoethanol per 1 mL RB Buffer. This mixture can be stored for 4 weeks at room temperature.

Preparing Reagents

• Dilute RNA Wash Buffer II with 100% ethanol as follows and store at room temperature.

Kit	100% Ethanol to be Added
R6827-00	20 mL
R6827-01	100 mL
R6827-02	200 mL

 

• Add 20 µL 2-mercaptoethanol per 1 mL RB Buffer. This mixture can be stored for 4 weeks at room temperature.

Note: Only prepare what is needed. RB Buffer is required without the addition of 2-mercaptoethanol in the Difficult Sample Types protocol.

• For Difficult Sample Types protocol (Page 12), add 20 µL 2-mercaptoethanol per 1 mL NTL Lysis Buffer. This mixture can be stored for one month at room temperature.

Quantification of RNA

Quantification and Storage of RNA

To determine the concentration and purity of RNA, measure absorbance at 260 nm and 280 nm with a spectrophotometer. One OD unit measured at 260 nm corresponds to 40 μg/mL RNA. Nuclease-free Water is slightly acidic and can dramatically lower absorbance values. We suggest that you dilute the sample in a buffered solution (TE) for spectrophotometric analysis. The A260/A280 ratio of pure nucleic acids is 2.0, while an A260/A280 ratio of 0.6 denotes pure protein. A ratio of 1.8-2.0 corresponds to 90%-100% pure nucleic acid. Phenol has a maximum absorbance at 270 nm and can interfere with spectrophotometric analysis of DNA or RNA. However, the E.Z.N.A.® Plant RNA Kit eliminates the use of phenol and avoids this problem. Store RNA samples at -70°C in water. Under these conditions, RNA is stable for more than a year.

Integrity of RNA

It is highly recommended that RNA quality be determined prior to beginning all downstream applications. The quality of RNA can be best assessed by denaturing agarose gel electrophoresis with ethidium bromide staining. The ribosomal RNA bands should appear as sharp, clear bands on the gel. The 28S band should appear to be double that of the 18S RNA band (23S and 16S if using bacteria). If the ribosomal RNA bands in any given lane are not sharp and appear to be smeared towards the smaller sized RNA, it is very likely that the RNA undergone degradation during the isolation, handling, or storage procedure. Although RNA molecules less than 200 bases in length do not efficiently bind to the HiBind® matrix, a third RNA band, the tRNA band, may be visible when a large number of cells are used.

Expected Yields

Sample yields from 100 mg starting tissue.

Yields obtained with the E.Z.N.A.® Plant RNA Kit
Arabidopsis	30 μg
Maize Leaves	65 μg
Mustard Leaves	34 μg
Tobacco Leaves	28 μg

E.Z.N.A.® Plant RNA Kit Standard Protocol

E.Z.N.A.® Plant RNA Kit - Standard Protocol

This protocol is suitable for most fresh or frozen tissue samples, thereby allowing efficient recovery of RNA. However, due to the tremendous variation in water and polysaccharide content in plants, sample size should be limited to ≤100 mg. Best results are obtained with young leaves or needles. The method outlined in this protocol will isolate a sufficient amount of RNA for tracks on a standard Northern assay.

Materials and Equipment to be Supplied by User:

• Microcentrifuge capable of at least 14,000 x g

• RNase-free pipet tips and 1.5 mL microcentrifuge tubes

• 100% ethanol • 70% ethanol

• 2-mercaptoethanol (14.2 M)

• Liquid nitrogen

• Disposable pestles

• Optional: Water bath, incubator, or heat block capable of 65°C

Before Starting:

• Prepare RNA Wash Buffer ll and RB Buffer according to “Preparing Reagents” section on Page 6

 

1. Collect tissue in a 1.5 mL microcentrifuge tube (not provided) and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube. Grind the tissue using disposable pestles.

Note: Disposable Kontes pestles work well and are available for purchase (product no. SSI-1014-39 & SSI-1015-39). One can allow liquid nitrogen to evaporate and store the samples at -70°C for later use. Do not allow samples to thaw. Use disposable pestle only once. Alternatively, a small clean mortar and pestle can be used. The above methods for disrupting plant tissue cannot be replaced with mechanical homogenizers. 

2. Transfer up to 100 mg frozen ground plant tissue to a new 1.5 mL microcentrifuge tube.

Note: We recommend starting with 50 mg of tissue at first. If results obtained are satisfactory, you may start increasing the amount of starting material. Samples should not be allowed to thaw before the addition of RB Buffer in Step 3.

3. Immediately add 500 μL RB Buffer. Vortex at maximum speed to mix thoroughly.

Note: RB Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 6 for instructions. Ensure that all the samples are completely suspended and that there are no clumps in the solution. Clumps will result in low yields.

4. Insert a Homogenizer Mini Column into a 2 mL Collection Tube.

5. Transfer the lysate to a Homogenizer Mini Column.

6. Centrifuge at 14,000 x g for 5 minutes at room temperature.

7. Transfer cleared lysate to a new 1.5 mL microcentrifuge tube. Do not disturb or transfer any of the insoluble pellet. Measure the volume of the lysate.

8. Add 1 volume 70% ethanol. Vortex at maximum speed for 20 seconds. A precipitate may form at this point; it will not interfere with DNA isolation. Passing the mixture through a needle using a syringe or by pipetting up and down 10-15 times may break up the precipitates.

9. Insert a HiBind® RNA Mini Column into a 2 mL Collection Tube.

10. Transfer 700 µL sample, including any precipitates that may have formed, to the HiBind® RNA Mini Column.

11. Centrifuge at 12,000 x g for 1 minute at room temperature.

12. Discard filtrate and reuse the collection tube.

13. Repeat Steps 10-12 until all of the sample has been transferred to the column.

E.Z.N.A.® Plant RNA Kit Standard Protocol

Optional: This the starting point of the optional on-membrane DNase I Digestion Protocol. Since the HiBind® matrix of the RNA Mini Column eliminates most DNA, DNase I digestion is not necessary for most downstream applications. However, certain sensitive RNA applications may require further DNA removal. If an additional RNA removal step is required, please continue to the DNase I Digestion Protocol found on Page 19. (See DNase I Digestion Set, Cat # E1091 for more information). If DNase I digestion is not required, proceed to Step 14.

14. Add 500 μL RNA Wash Buffer I.

15. Centrifuge at 10,000 x g for 30 seconds.

16. Discard the filtrate and the collection tube.

17. Transfer the HiBind® RNA Mini Column to a new 2 mL Collection Tube.

18. Add 700 μL RNA Wash Buffer II.

Note: RNA Wash Buffer II must be diluted with 100% ethanol before use. Please see Page 6 for instructions.

19. Centrifuge at 10,000 x g for 30 seconds.

20. Discard the filtrate and reuse collection tube.

21. Add 500 μL RNA Wash Buffer II.

22. Centrifuge at 10,000 x g for 30 seconds.

23. Discard the filtrate and reuse collection tube.

24. Centrifuge the empty HiBind® RNA Mini Column at maximum speed for 2 minutes to dry the column.

Note: This step is critical for removal of trace ethanol that may interfere with downstream applications.

25. Transfer the HiBind® RNA Mini Column to a clean 1.5 mL microcentrifuge tube.

26. Add 50-100 μL Nuclease-free Water. Note: Make sure to add water directly onto the HiBind® RNA Mini Column matrix.

27. Centrifuge at maximum speed for 1 minute and store eluted RNA at -70°C.

Note: Any combination of the following steps can be used to help increase RNA yield.

• Heat the Nuclease-free Water to 65°C before adding to the column.

• Let sit at room temperature for 5 minutes.

• Increase the elution volume.

• Repeat the elution step with fresh Nuclease-free Water (this may increase the yield, but decrease the concentration).

• Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).

E.Z.N.A.® Plant RNA Kit Difficult Samples Protocol

E.Z.N.A® Plant RNA Kit - Difficult Sample Types

In certain plant samples, RNA isolation can be difficult due to their large amount of polysaccharides and phenolic compounds. This protocol involves a simple and rapid precipitation that will remove much of these compounds. Use this protocol when the standard protocol (Page 8) results in low RNA yields.

Materials and Equipment to be Supplied by User:

• Microcentrifuge capable of at least 14,000 x g

• RNase-free pipet tips and 1.5 mL microcentrifuge tubes

• Water bath, incubator, or heat block capable of 65°C

• 100% ethanol

• 70% ethanol

• 100% isopropanol

• 2-mercaptoethanol (14.2 M)

• Liquid nitrogen

Before Starting:

• Prepare RNA Wash Buffer ll, RB Buffer, and NTL Lysis Buffer according to “Preparing Reagents” section on Page 6

• Set water bath, incubator, or heat block to 65°C

• Heat RB Buffer or Nuclease-free Water to 65°C (whichever applicable, see Step 11; RB Buffer is recommended)

1. Collect tissue in a 1.5 mL microcentrifuge tube (not provided) and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube. Grind the tissue using disposable pestles.

Note: Disposable Kontes pestles work well and are available for purchase (product no. SSI-1014-39 & SSI-1015-39). One can allow liquid nitrogen to evaporate and store the samples at -70°C for later use. Do not allow samples to thaw. Use disposable pestle only once. Alternatively, a small clean mortar and pestle can be used. The above methods for disrupting plant tissue cannot be replaced with mechanical homogenizers.

2. Transfer up to 100 mg frozen ground plant tissue to a new 1.5 mL microcentrifuge tube.

Note: We recommend starting with 50 mg of tissue at first. If results obtained are satisfactory, you may start increasing the amount of starting material. Samples should not be allowed to thaw before the addition of NTL Lysis Buffer in Step 3.

3. Immediately add 600 μL NTL Lysis Buffer. Vortex at maximum speed to mix thoroughly.

Note: NTL Lysis Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 6 for instructions. Ensure that all the samples are completely suspended and that there are no clumps in the solution. Clumps will result in low yields.

4. Add 140 µL SP Buffer. Vortex to mix thoroughly.

5. Centrifuge at 10,000 x g for 10 minutes.

6. Transfer cleared lysate to a new 1.5 mL microcentrifuge tube. Do not disturb or transfer any of the insoluble pellet. Measure the volume of the lysate.

7. Add 1 volume isopropanol. Vortex to mix thoroughly.

Note: In most cases 600 μL cleared lysate can easily be removed. Add 600 μL isopropanol. Depending on the sample, the volume of the cleared lysate will vary. This step removes much of the polysaccharide content and improves spin-column performance by increasing RNA binding capacity (and therefore yield) in the steps that follow. Incubation is not required after the addition of isopropanol.

8. Immediately centrifuge at 10,000 x g for 2 minutes. Longer centrifugation does not improve yield.

9. Carefully aspirate or decant the supernatant. Do not disturb the RNA pellet. Invert the microcentrifuge tube on a paper towel for 1 minute to allow any residual liquid to drain. Drying the pellet is not necessary.

10. Add 100 μL RB Buffer or sterile Nuclease-free Water heated to 65°C. Vortex at maximum speed to resuspend the pellet. A brief incubation at 65°C may be necessary to effectively dissolve the RNA.

Important: Do not add 2-mercaptoethanol to RB Buffer for this step. RB Buffer is recommended for dissolving the RNA pellet, especially when degradation was found after elution. RB Buffer contains a strong RNase inhibitor. Nuclease-free Water should only be used when dissolving the RNA when RB Buffer has proven difficult.

11. Adjust the binding conditions by following either A or B below.

Note: RB Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 6 for instructions.

A. If RB Buffer was used in Step 10, add 250 µL RB Buffer and 350 µL 100% ethanol.

B. If Nuclease-free Water was used in Step 10, add 350 µL RB Buffer and 250 µL 100% ethanol.

12. Insert a HiBind® RNA Mini Column into a 2 mL Collection Tube.

13. Transfer the entire sample (including any precipitate that may have formed) to the HiBind® RNA Mini Column.

14. Centrifuge at 10,000 x g for 30 seconds.

15. Discard the filtrate and reuse the Collection Tube.

Optional: This the starting point of the optional on-membrane DNase I Digestion Protocol. Since the HiBind® matrix of the RNA Mini Column eliminates most DNA, DNase I digestion is not necessary for most downstream applications. However, certain sensitive RNA applications may require further DNA removal. If an additional RNA removal step is required, please continue to the DNase I Digestion Protocol found on Page 19. (See DNase I Digestion Set, Cat # E1091 for more information). If DNase I digestion is not required, proceed to Step 16.

16. Add 500 μL RNA Wash Buffer I.

17. Centrifuge at 10,000 x g for 30 seconds.

18. Discard the filtrate and the collection tube.

19. Transfer the HiBind® RNA Mini Column to a new 2 mL Collection Tube.

20. Add 700 μL RNA Wash Buffer II.

Note: RNA Wash Buffer II must be diluted with 100% ethanol before use. Please see Page 6 for instructions.

21. Centrifuge at 10,000 x g for 30 seconds.

22. Discard the filtrate and reuse collection tube.

23. Add 500 μL RNA Wash Buffer II.

24. Centrifuge at 10,000 x g for 30 seconds.

25. Discard the filtrate and reuse collection tube.

26. Centrifuge at maximum speed for 1 minute to completely dry the HiBind® RNA Mini Column.

Note: It is important to dry the HiBind® RNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.

27. Transfer the HiBind® RNA Mini Column to a clean 1.5 mL microcentrifuge tube.

28. Add 50-100 μL Nuclease-free Water.

Note: Make sure to add water directly onto the HiBind® RNA Mini Column matrix.

29. Centrifuge at maximum speed for 1 minute and store eluted RNA at -70°C.

Note: Any combination of the following steps can be used to help increase RNA yield.

• Heat the Nuclease-free Water to 65°C before adding to the column.

• Let sit at room temperature for 5 minutes.

• Increase the elution volume.

• Repeat the elution step with fresh Nuclease-free Water (this may increase the yield, but decrease the concentration).

• Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).

E.Z.N.A.® Plant RNA Kit Arthropod Samples Protocol

E.Z.N.A® Plant RNA Kit - Arthropod Samples

The exoskeleton of arthropods poses the same problems that occur when trying to isolate RNA from plant specimens. Pigments and polysaccharides often co-purify with nucleic acids, and interfere with downstream applications.

Materials and Equipment to be Supplied by User:

• Microcentrifuge capable of at least 14,000 x g

• RNase-free pipet tips and 1.5 mL microcentrifuge tubes

• Water bath, incubator, or heat block capable of 65°C

• 100% ethanol

• 70% ethanol

• 2-mercaptoethanol (14.2 M)

• Liquid nitrogen

Before Starting:

• Prepare RNA Wash Buffer ll and RB Buffer according to “Preparing Reagents” section on Page 6

• Set water bath, incubator, or heat block to 65°C

1. Freeze and grind up to 100 mg arthropod tissue in liquid nitrogen. Grind tissue completely to obtain a fine homogenous powder.

2. Immediately add 500 μL RB Buffer. Vortex at maximum speed to mix thoroughly.

Note: RB Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 6 for instructions. Ensure that all the samples are completely suspended and that there are no clumps in the solution. Clumps will result in low yields.

3. Proceed Step 4 of the Standard Protocol (Page 9).

E.Z.N.A.® Plant RNA Kit Fungal Samples Protocol

E.Z.N.A® Plant RNA Kit - Fungal Samples

The E.Z.N.A.® Plant RNA Kit can also be used for fungal RNA isolation, since many fungal samples posses similar cellular attributes to plant specimens.

Materials and Equipment to be Supplied by User:

• Microcentrifuge capable of at least 14,000 x g

• RNase-free pipet tips and 1.5 mL microcentrifuge tubes

• Water bath, incubator, or heat block capable of 65°C

• 100% ethanol

• 70% ethanol

• 2-mercaptoethanol (14.2 M)

• Liquid nitrogen

Before Starting:

• Prepare RNA Wash Buffer ll and RB Buffer according to “Preparing Reagents” section on Page 6

• Set water bath, incubator, or heat block to 65°C

1. Freeze and grind up to 30 mg fungal tissue in liquid nitrogen. Grind tissue completely to obtain a fine homogenous powder.

2. Immediately add 500 μL RB Buffer. Vortex at maximum speed to mix thoroughly.

Note: RB Buffer must be mixed with 2-mercaptoethanol before use. Please see Page 6 for instructions. Ensure that all the samples are completely suspended and that there are no clumps in the solution. Clumps will result in low yields.

3. Proceed Step 4 of the Standard Protocol (Page 9).

E.Z.N.A.® Plant RNA Kit DNase I Digestion Protocol

E.Z.N.A.® Total RNA Kit I - DNase I Digestion Protocol

Since the HiBind® matrix of the RNA Mini Column eliminates most DNA, DNase I digestion is not necessary for most downstream applications. However, certain sensitive RNA applications may require further DNA removal. (See DNase I Digestion Set, Cat# E1091 for further information).

After completing Steps 1-13 of the Standard Protocol (Pages 8-9) or Steps 1-15 of the Difficult Tissue Samples Protocol (Pages 12-14), proceed with the following protocol.

User Supplied Material:

• DNase I Digestion Set (E1091)

1. For each HiBind® RNA Mini Column, prepare the DNase I stock solution as follows:

Buffer	Volume per Prep
E.Z.N.A.® DNase I Digestion Buffer	73.5 μL
RNase-free DNase I (20 Kunitz/µL)	1.5 μL
Total Volume	75 μL

Important Notes:

• DNase I is very sensitive and prone to physical denaturing. Do not vortex the DNase I mixture. Mix gently by inverting the tube.

• Freshly prepare DNase I stock solution right before RNA isolation.

• Standard DNase buffers are not compatible with on-membrane DNase I digestion. The use of other buffers may affect the binding of RNA to the HiBind® matrix and may reduce RNA yields and purity.

• All steps must be carried out at room temperature. Work quickly, but carefully.

2. Insert the HiBind® RNA Mini Column containing the sample into a 2 mL Collection Tube

3. Add 250 µL RNA Wash Buffer I to the HiBind® RNA Mini Column.

4. Centrifuge at 10,000 x g for 1 minute.

5. Discard the filtrate and reuse the Collection Tube.

6. Add 75 μL DNase I digestion mixture directly onto the surface of the membrane of the HiBind® RNA Mini Column.

Note: Pipet the DNase I directly onto the membrane. DNA digestion will not be complete if some of the mixture is retained on the wall of the HiBind® RNA Mini Column.

7. Let sit at room temperature for 15 minutes.

8. Add 250 μL RNA Wash Buffer I to the HiBind® RNA Mini Column.

9. Let sit at room temperature for 2 minutes.

10. Centrifuge at 10,000 x g for 1 minute.

11. Discard the filtrate and reuse the Collection Tube.

12. Add 600 μL RNA Wash Buffer II.

Note: RNA Wash Buffer II must be diluted with 100% ethanol before use. Please see Page 6 for instructions.

13. Centrifuge at 10,000 x g for 1 minute.

14. Discard the filtrate and reuse the Collection Tube.

15. Repeat Steps 12-14 for a second RNA Wash Buffer II wash step.

16. Centrifuge at maximum speed for 2 minutes to completely dry the HiBind® RNA Mini Column matrix.

Note: It is important to dry the HiBind® RNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.

17. Place the column in a clean 1.5 mL microcentrifuge tube (not supplied).

18. Add 50-100 μL Nuclease-free Water.

Note: Make sure to add water directly onto the HiBind® RNA Mini Column matrix.

19. Centrifuge at maximum speed for 2 minutes and store eluted RNA at -70°C.

Note: Any combination of the following steps can be used to help increase RNA yield.

• Heat the Nuclease-free Water to 65°C before adding to the column.

• Let sit at room temperature for 5 minutes.

• Increase the elution volume.

• Repeat the elution step with fresh Nuclease-free Water (this may increase the yield, but decrease the concentration).

• Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).

Troubleshooting Guide

Please use this guide to toubleshoot any problems that may arise. For further assistance, please contact the technical support staff , toll free, at 1-800-832-8896.

Possible Problems and Suggestions

Problem	Cause	Solution
Little or no RNA eluted	RNA remains on the column	Repeat elution step.
		Heat Nuclease-free Water to 65°C prior to elution.
	Column is overloaded	Reduce quantity of starting material.
Problem	Cause	Solution
Clogged column	Incomplete homogenization	Completely homogenize sample
		Increase centrifugation time
		Reduce amount of starting material
Problem	Cause	Solution
Degraded RNA	Starting sample problems	Freeze starting material quickly in liquid nitrogen.
		Follow protocol closely, and work quickly
	RNase contamination	Ensure not to introduce RNase during the procedure.
		Check buffers for RNase contamination.
Problem	Cause	Solution
Problem in downstream applications	Salt carryover during elution	Ensure RNA Wash Buffer II has been diluted with 4 volumes 100% ethanol as indicated on bottle.
		RNA Wash Buffer II must be stored and used at room temperature.
		Repeat wash with RNA Wash Buffer II.
Problem	Cause	Solution
DNA contamination	DNA contamination	Perform the optional DNase I Digestion Protocol on Page 19.
Problem	Cause	Solution
Low Abs ratios	RNA diluted in acidic buffer or water	Nuclease-free Water is acidic and can dramatically lower Abs260 values. Use TE buffer to dilute RNA prior to spectrophotometric analysis.

Ordering Information

The following components are available for purchase separately.

(Call Toll Free 1-800-832-8896)

Product	Part Number
RB Buffer, 100 mL	PR026
RNA Wash Buffer I, 100 mL	PR030
RNA Wash Buffer II, 25 mL	PR031
2 mL Collection Tubes, 500/pk	SSI-1370-00-01
2 mL Collection Tubes, 500/pk, 50 pk/cs	SSI-1370-00
1.5 mL DNase/RNase-free Microcentrifuge Tubes, 500/pk, 10 pk/cs	SS1-1210-00
RNase-free DNase Set, 50 preps	E1091
RNase-free DNase Set, 200 preps	E1091-02
Homogenizer Mini Columns, 200 preps	HCR003
Proteinase K (>600 mAU/mL, Solution), 2 mL	AC115
Proteinase K (>600 mAU/mL, Solution), 10 mL	AC116

  

 

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