Download Nucleic Acid Purification From Plant Tissues for Viral Detection

Nucleic Acid Purification From Plant Tissues for Viral Detection
Samantha Lewis1, Laurence Delaurière2, Becky Carroll1, Elisa Ruiz2, Ellyn Lepinski1, Giuseppe Durante3,
Caroline Chatillon4, Doug Wieczorek1,Céline Ménager2, Eric B. Vincent1 and Trista Schagat1
1Promega
Corporation, 2800 Woods Hollow, Madison WI, USA 53711, 2Promega Europe Training and Applications Laboratory (PETAL), 24 Chemin des Verrières,
Charbonnières-les-Bains, France 69260, 3International Plant Analysis and Diagnostics srl., Loc. Cascina Codazza, Via Einstein, Italy 26900, 4Qualiplante, Cap Alpha,
Avenue de l'Europe, Clapiers, France 34830.
1. Introduction
Results:
7. RNA Isolation from Virus in Plant
Tissues
Viral RNA Isolation From Seeds
DNA Concentration
50
Concentration (ng/µl)
Concentration of DNA isolated from
20mg of liquid nitrogen ground leaf
tissue, measured by QuantiFluor® ONE
dsDNA System.
Methods: Spike Tobacco Mosaic Virus into seed lysates and purify RNA
using the Maxwell® RSC Plant RNA Kit for viral detection with RT-qPCR.
40
30
20
Sample: Three species of plant seeds
10
0
A260/A280
2.5
Soy
Cotton
A260/A230
Tomato Seed
Pepper Seed
2.0
Absorbance at 260, 230, and 280nm
was read by NanoDrop. Excellent
absorbance ratios (greater than 1.8)
were observed for all samples.
1.5
1.0
0.5
2. Methods: A Complete Nucleic Acid
Toolbox for Plant Samples
Soy
Total RNA Concentration and Purity
NANOVUE
• Concentrated, pure
RNA isolation from
plant seeds
0.0
Corn
Results:
Cotton
qPCR Ct
23
qPCR was run with GoTaq® qPCR
Master Mix with plant species specific
primers.
Ct
Flexible, effective workflow options for nucleic acid purification,
quantitation, and amplification from botanical samples. Tested
for a variety of plant types and tissues.
• Excellent >80%
recovery of spiked
Tobacco Mosaic Virus
RNA
21
19
17
Soy
2.0
300
1.5
200
1.0
100
0.5
0
0.0
35
30
Sample Type % Viral Recovery
Pepper
119.8
Tomato
85.0
Watermelon
124.5
Results:
Concentration (ng/µl)
Concentration of RNA isolated from
20mg of liquid nitrogen ground leaf
tissue, measured by QuantiFluor® RNA
System.
10
8
6
A260/A280
5
0
2
Corn
Soy
Results:
A260/A230
Absorbance Ratio
2
Absorbance at 260, 230, and 280nm
was read by NanoDrop. Excellent
absorbance ratios (greater than 1.8)
were observed for all samples.
1.5
1
0.5
0
Corn
Soy
GoTaq® Systems
ΔCt
ReliaPrep™ Manual Nucleic Acid Purification
Systems for Plant Samples
Soy
Average Ct
qPCR with DNA from wood
1:10
S. Tuberosum tubers
Elute
Average Ct
RT-qPCR with RNA from 10mg potato tissue
Sample: Three species of plant leaves
27
26
25
24
23
22
21
20
Soybean Leaf
DNA from meristem and root
Theobroma cacao meristem and root
Integrity of DNA from Cacao tissue
Root
Meristem
28
24
22
Infected Infected Infected Infected Infected Negative
Leaf 1 Leaf 2 Leaf 3 Leaf 4 Leaf 5
Leaf
Melt Curve of Controls
Melt Curve of Infected
Leaf RNA
• RNA isolated from
infected leaf samples
produced specific melt
curves with the CEVd
assay.
Negative
20mg 75mg 150mg
9. Summary
Conclusions:
• Nucleic acid can be isolated from plants in three flexible formats:
o Manual/low throughput: ReliaPrep™ Miniprep Systems
o Low/medium throughput: Maxwell® RSC Instrument and Kits
o High throughput: Maxwell® HT
• Downstream quantitation and amplification products compliment this
plant based workflow:
o QuantiFluor® fluorescent dye-based nucleic acid quantification
o GoTaq® amplificaiton reagents
• Nucleic acid isolation from a number of plant leaves, branches,
meristems, roots, beans, and seeds have been tested with Maxwell®
and Reliaprep™ Systems
RNA from beans
Coffea arabica and robusta beans
Integrity of RNA from Coffea tissue
arabica
robusta
• Viral RNA from infected leaves and seeds can also be isolated
Resources:
Sample Nucleic Acid
DNA
RNA
Leaf
miRNA
DNA
RNA
Seed
miRNA
Purification Solutions for Common Plant Tissue Types
Reliaprep™ System
Reliaprep™ gDNA Tissue Miniprep System
Reliaprep™ RNA Tissue Miniprep System
Reliaprep™ miRNA Miniprep System
Reliaprep™ gDNA Tissue Miniprep System
Reliaprep™ RNA Tissue Miniprep System
Reliaprep™ miRNA Miniprep System
Maxwell® RSC
Maxwell® RSC Plant DNA Kit
Maxwell® RSC Plant RNA Kit
Maxwell® RSC miRNA Kit
Maxwell® RSC Plant DNA Kit
Maxwell® RSC Plant RNA Kit
Maxwell® RSC miRNA Kit
Maxwell® HT
Custom
Options
Available
* Due to the diversity present in plants these are suggestions, and results may vary. Contact Promega Technical Services with any questions
Red
Corn Leaf
30
Cotton
1:100
RNA from tubers
Wash
32
Positive
20mg
Neat
Negative Control
Cotton
DNA from branch tissue
Method: A Simple Spin-Column Based Workflow
Lysis
Soy
We have optimized a number of protocols to obtain amplifiable
nucleic acid from difficult and diverse plant tissues.
26
24
22
20
18
16
14
12
10
Series1
1:100
100mg of Prunus sp. branch tissue
RNA: ReliaPrep™ RNA Tissue Miniprep System
DNA: ReliaPrep™ gDNA Tissue Miniprep System
Corn
6. Nucleic Acid Extraction from Diverse
Botanical Tissue Types
3. A Manual Nucleic Acid Isolation From
Three Species of Plant Leaves
• Amplifiable CEVd
Viroid RNA was
isolated from all
infected leaf samples.
• RNA isolated from a
CEVd negative leaf
did not amplify below
negative control Ct
values.
RT-qPCR inhibition was estimated by
calculating the ΔCT between neat and
1:10 and 1:100 diluted samples (no
inhibition should be 3.3).
Corn
Add to
Column
29
27
25
23
21
19
17
15
RT-qPCR Inhibition
Assay flexibility:
• qPCR or RT-qPCR
• 1-Step or 2-Step RT
• Probe or dye
• Fast or standard cycling
34
26
Ct
Amplification was run with GoTaq® 1Step RT-qPCR System with plant
species specific primers.
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Watermelon
Viroid RNA amplification by CEVd specific RT-qPCR
Cotton
RT-qPCR Ct
1:10
Tomato
Cotton
2.5
Amplify:
10
Sample: Leaves from infected and healthy lemon
trees, kindly provided by International Plant
Analysis and Diagnostics Srl.
4
Purity
Dye Based Quantitation
• High sensitivity
• Accurate
• dsDNA, ssDNA, or RNA
15
Methods: Extraction of Citrus exocortis viroid (CEVd) RNA from infected
lemon leaves for viral detection with Qualiplante CEVd One-Step RealTime RT-PCR Assay. The small size of this viroid RNA requires use of
the Maxwell RSC miRNA Kit for best viral recovery.
RNA Concentration
12
QuantiFluor® System
20
8. Viroid RNA Extraction From Citrus
Leaves
0
Quantify:
25
Cotton
5. Manual RNA Isolation From Three
Species of Plant Leaves
For protocols and example data for a number of diverse plant types visit
the applications selector at Promega.com.
TMV Yield (ng)
1:100
Soy
Watermelon
Viral RNA quantification by RT-qPCR
Pepper
Corn
Find Your Plant Type using the Applications
Selector Tool
Tomato
Cotton
qPCR inhibition was estimated by
calculating the ΔCT between neat and
1:10 and 1:100 diluted samples (no
inhibition should be 3.3).
• ReliaPrep™ MiniPreps
• Maxwell® RSC
• High throughput options
A260/A230
400
Pepper
Average Ct
ΔCt
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
A260/A280
2.5
qPCR Inhibition
1:10
QUANTUS
500
15
Corn
Watermelon Seed
Absorbance Ratio
Corn
Purity
Total RNA Concentration
(ng/µl)
The diverse range of plant species and tissue types found in
nature present a unique set of obstacles for nucleic acid
isolation from botanical samples. In addition, the carry-over of
substances present in certain plants can interfere with
quantitation and downstream amplifications. These difficulties
highlight the need for robust, reliable chemistries for molecular
biology studies with plants. We present several solutions to
purify and amplify nucleic acid from a variety of plant types
and tissues with a focus on agriculturally relevant applications.
Absorbance Ratio
`
4. Manual DNA Isolation From Three
Species of Plant Leaves
Russet
Cotton Leaf
For additional Maxwell® or ReliaPrep™ information, visit:
• Maxwell® : http://www.promega.com/products/instruments/maxwell-systems/
www.promega.com
• ReliaPrep™: http://www.promega.com/products/pm/rna-purification-from-cellsand-tissues/