Pospiviroid RT-PCR Detection Kit - Protocol
Contents
1. Table 1 RT PCR Assay Preparation RT PCR Components Volume Per RT PCR Reaction Sample RNA 2 uL Nuclease Free Water 8 uL Total Volume 20 uL 2 For every RT PCR run prepare one positive control RT PCR as shown in Table 2 below Table 2 RT PCR Positive Control Preparation RT PCR Components Volume Per RT PCR Reaction Total Volume 20 uL 3 For every RT PCR run prepare one no template control RT PCR as shown in Table 3 below Table 3 RT PCR Negative Control Preparation RT PCR Components Volume Per RT PCR Reaction Nuclease Free Water 10 pL Total Volume 20 uL Therefore at a minimum each PCR run will contain 6 separate RT PCR reactions C Pospiviroid One Step RT PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step RT PCR Table 4 Pospiviroid Assay Program One Step RT PCR Cycle Step Temperature Duration Cycle 1 Step 1 50 C 30 min Cycle 2 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 3 40x Step 2 55 C 30 sec Step 3 72 C 45 sec Cycle 4 Step 1 72 C 5 min Cycle 5 Step 1 4 C 0 D Pospiviroid One Step RT PCR Assay Results Interpretation For the analysis of the RT PCR data the entire 15 20 uL RT PCR Reaction should be loaded on a 1X TAE 1 5 Agarose RNA gel along with 10 uL of Norgen s RNA Marker provided 2 The RT PCR products should b2. 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 or call one of the NORGEN local distributors www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2014 Norgen Biotek Corp PI53700 2
3. are recommended as your best defense against these enzymes The RNA area should be located away from microbiological work stations Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is recommended that gloves are changed frequently to avoid contamination There should be designated solutions tips tubes lab coats pipettes etc for RNA only All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water Clean all surfaces with commercially available RNase decontamination solutions When working with purified RNA samples ensure that they remain on ice during downstream applications Protocol A Pospiviroid Total RNA Isolation Important Notes Prior to Beginning Protocol All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Wash Solution by adding 25 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will
4. entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute Depending on your lysate volume repeat step 2c if necessary 3 Column Wash a Apply 400 uL of Wash Solution to the column and centrifuge for 1 minute 9 Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute Discard the flowthrough and reassemble the spin column with its collection tube Repeat steps 3a and 3b to wash column a second time Wash column a third time by adding another 400 uL of Wash Solution and centrifuging for 1 minute Discard the flowthrough and reassemble the spin column with its collection tube Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 4 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 uL of Elution Buffer to the column c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by a1 minute spin at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute d The purified RNA sample could be used immediately for RT PCR as described below It is recommended that samples be placed at 70 C for l
5. give a final volume of 36 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added Both fresh or frozen samples may be used for this procedure Samples should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Do not allow frozen samples to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised While the provided procedure does not rely on the use of liquid nitrogen to homogenize the sample both fresh and frozen tissues can optionally be processed using other homogenization methods including grinding with liquid nitrogen Isolation Control soC An Isolation Control soC is supplied This allows the user to control the RNA isolation procedure For this assay add the Isolation Control soC to the lysate during the isolation procedure The Isolation Control IsoC must not be added to the sample material directly Do not freeze and thaw the Isolation Control IsoC more than 2 times The Pospiviroid Isolation Control IsoC must be kept on ice at all times during the isolation procedure The RT PCR components of the Pospiviroid RT PCR Detection Kit should remain at 20 C until RNA is extracted and ready for RT PCR amplification It is important to work quickly during this procedure 1 Lysate Preparation a Transfer lt 100 mg of plant tissue into a mortar that contai
6. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK ee CORPORATION Email techsupport norgenbiotek com Pospiviroid RT PCR Detection Kit Product Insert Product 53700 Pathogen Information Pospiviroid belongs to the Pospiviroidae family on the basis of biochemical biological and structural properties which have been classified The genus Pospiviroid contains nine viroid species Potato spindle tuber viroid PSTVd Tomato chlorotic dwarf viroid TCDVd Mexican papito viroid MPVd Tomato planta macho viroid TPMVd Chrysanthemum stunt viroid CSVd Citrus exocortis viroid CEVd Tomato apical stunt viroid TASVd Iresine viroid IrVd and Columnea latent viroid CLVd Viroids cannot be detected by immunological methods because they do not encode any proteins therefore an accurate and reliable diagnostic method for the detection of viroids followed by eradication of the infected sources is needed for quarantine operations Norgen s Pospiviroid Viroid RT PCR Detection Kit allows for the detection of any of the 9 viroid species of the Pospiviroid family Principle of the Test Norgen s Pospiviroid Viroid RT PCR Detection Kit constituents a ready to use system for the isolation and detection of Pospiviroid using end point one step RT PCR The kit first allows for the isolation of viroid RNA from plant tissues using spin column chromatography based on Norgen s pr
7. action Conirol Reaction Interpretation Pospiviroid Target IsoC Band PCRC Band Band 184 bp 499 bp 150 bp Positive Control X X X Valid Negative Control X Valid Sample X X Positive Sample xX Negative Sample X Re test Sample Re test Sample X Negative Sample X X Positive Sample X X Positive Sample X Re test For results obtained that are not covered in Table 5 above please refer to the Troubleshooting Section E Pospiviroid RT PCR Assay Specificity and Sensitivity e The specificity of Norgen s Pospiviroid RT PCR Detection Kit is first and foremost ensured by the selection of the Pospiviroid specific primers as well as the selection of stringent reaction conditions The Pospiviroid universal primers were checked for possible homologies to all plant viroids in GenBank and published Pospiviroid sequences by sequence comparison analysis e Pospiviroid specificity Pospiviroid Specificity Viroid Detection In vivo Primer specificity In Silico Potato spindle tuber viroid PSTVd Tomato chlorotic dwarf viroid TCDVd S Tomato apical stunt viroid TASVd NT Tomato planta macho viroid TPMVd Mexican papita Viroid MPVd NT Chrysanthemum stunt viroid CSVd Iresine viroid 1 IrVd 1 NT Citrus exocortis viroid CEVd 4 Colummnea latent viroid CLVd NT NT Not Tested In vitro transcribed RNA was used with an in vivo one step RT PCR assay Base
8. d on NCBI sequencing BLAST search result F Linear Range e The linear range of Norgen s Pospiviroid RT PCR Detection Kit was determined by analysing a dilution series of a Citrus Exocortis Viroid CEVd quantification standards ranging from 100 ag to 1 pg e Each dilution has been tested in replicates n 4 using Norgen s Pospiviroid RT PCR Detection Kit on a 1X TAE 1 5 agarose gel e The linear range of Norgen s Pospiviroid RT PCR Detection Kit has been determined to cover concentrations from 100 ag to 1 ng e Under the conditions of the Norgen s Pospiviroid RNA Isolation procedure Norgen s Pospiviroid RT PCR Detection Kit covers a linear range from 100 copies to 1 x 10 copies Frequently Asked Questions 1 How many samples should be included per RT PCR run e Norgen s Pospiviroid RT PCR Detection Kit is designed to test 24 samples For every 6 samples a non template control Nuclease Free Water and a Positive Control must be included It is preferable to pool and test 6 samples at a time 2 How can interpret my results if neither the Pospiviroid RT PCR control nor the Isolation Control soC amplifies e f neither the Pospiviroid RT PCR control nor the Pospiviroid Isolation Control soC amplifies the sample must be re tested If the positive control showed amplification then the problem occurred during the isolation where as if the Positive control did not amplify therefore the problem has occurred during the
9. e resolved on the 1X TAE 1 5 Agarose gel at 150V for 20 minutes Gel running time will be vary depending on an electrophoresis apparatus 3 Sample results are provided below 1 2 3 4 5 6 7 8 E NTC CEVd 184bp fe 1 L I CETTE o Figure 1 Detection limit of Citrus Exocortis Viroid CEVd using the Pospivorid RT PCR Detection Kit A representative 1X TAE 1 4 agarose gel showing the amplification of CEVd at different concentrations 10 times serially diluted template used from 10 copies The size of the CEVd target amplicon corresponds to 184 bp NTC is the no template control Isolation control E E E E B PCR control L E P Figure 2 A representative 1X TAE 1 5 agarose gel showing the amplification of Isolation Control and PCR Control under different conditions using the Control 2X RT PCR Mastermix The size of the Isolation Control amplicon and PCR Control amplicon correspond to 499 bp and 150 bp respectively as represented by the provided DNA Marker M Lanes 1 to 6 showed detection of both Isolation Control and PCR Control suggesting that the RNA isolation as well as the RT PCR reaction was successful Lane 7 and 8 showed only the detection of PCR Control suggesting that while the RT PCR was successful the isolation failed to recover even the spiked in Isolation control Table 5 Interpretation of One Step RT PCR Assay Results Input Type Target re
10. er my third wash e Your first RNA elution will be contaminated with the Wash Solution This may dilute the RNA yield in your first elution and it may interfere with the PCR detection as ethanol is known to be a PCR inhibitor 10 What if forgot to add the Isolation Control soC during the isolation e It is recommended that the isolation is repeated Related Products Product Lysis Solution 100 mL 25806 Plant RNA DNA Purification Kit 24400 Plant Fungi RNA Purification Kit 25800 Viroid RNA Purification Kit 32800 Bacterial Genomic RNA Isolation Kit 17900 Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products If you have any questions or experience any difficulties regarding Norgen s Chrysanthemum Stunt Viroid Pospiviroid RT PCR Detection Kit or NORGEN products in general please do not hesitate to contact us NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at NORGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please contact our Technical Support Team between the hours of 8 30 and
11. ncluding the Pospi 2x RT PCR Master Mix Control 2X RT PCR Mastermix Isolation Control and Pospi Positive Control are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Pospiviroid RT PCR Detection Kit is designed for research purposes only Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual The customer must determine the suitability of the product for its particular use Disclaimers The Lysis Solution contains guanidinium salts and should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Safety Information Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com l CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Working with RNA RNases are very stable and robust enzymes that degrade RNA Autoclaving solutions and glassware is not always sufficient to actively remove these enzymes The first step when preparing to work with RNA is to create an RNase free environment The following precautions
12. ns 800 uL of Lysis Solution The volume of plant tissue and Lysis Solution can be increased proportionally For instance 0 5g of plant tissue requires 4 mL of Lysis Solution Extra Lysis Solution can be purchased separately See Related Products table Grind the sample using a pestle until the tissue is completely macerated Note Other homogenization devices such as Bioreba extraction bag and a homogenizer can also be applied to this procedure Using a pipette transfer the lysate into an RNAase free microcentrifuge tube not provided Spin the lysate for 2 minutes to pellet any cell debris Transfer the supernatant to another RNase free microcentrifuge tube Note the volume of the supernatant lysate Note Ensure that only the clear supernatant is transferred avoiding any of the debris If necessary repeat Step 1c if visible precipitates are still present after the first spin Add an equal volume of 96 100 ethanol provided by the user to the lysate collected above 100 uL of ethanol is added to every 100 uL of lysate Vortex to mix Proceed to Step 2 2 eee RNA to Column d Assemble a column with one of the provided collection tubes Add 10 uL of Isolation Control soC to the lysate mixture Apply up to 600 uL of the clarified lysate with ethanol onto the column and centrifuge for 1 minute at 14000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with the collection tube Note Ensure the
13. ong term storage B Pospiviroid RT PCR Assay Preparation Notes Before use suitable amounts of all RT PCR components should be completely thawed at room temperature gently vortexed and centrifuged briefly The amount of Pospiviroid 2X RT PCR Master Mix provided is enough for up to 32 RT PCR reactions 24 sample RT PCR 4 positive control RT PCR and 4 no template control RT PCR For each sample one RT PCR reaction using the Pospi 2X RT PCR Mastermix and one RT PCR reaction using Control 2X RT PCR Mastermix should be set up in order to have a proper interpretation of the results For every RT PCR run one reaction containing Pospiviroid Positive Control Pospi PosC and one reaction as no template control Nuclease Free Water must be included for proper interpretation of results e The recommended minimum number of RNA samples tested per RT PCR run is 6 e Using a lower volume from the sample than recommended may affect the sensitivity of Pospiviroid Limit of Detection 1 Prepare the RT PCR reaction for sample detection Set 1 using Pospi 2X RT PCR Mastermix and the RT PCR reaction for control detection Set 2 using Control 2X RT PCR Mastermix as shown in Table 1 below The recommended amount of sample RNA to be used is 1 2 uL Ensure that one Pospiviroid detection reaction and one control reaction is prepared for each RNA sample Adjust the final volume of the RT PCR reaction to 20 uL using the Nuclease Free Water provided
14. oprietary resin The RNA viroid is isolated free from inhibitors and can then be used as the template in a one step RT PCR reaction for Pospiviroid detection using the provided Pospi Master Mix The Pospiviroid Mastermix contains reagents and enzymes for the specific amplification of a 184 bp region of the viroid genome In addition Norgen s Pospiviroid RT PCR Detection Kit contains a second Mastermix the Control 2X RT PCR Master Mix which can be used to identify possible PCR inhibition and or inadequate isolation via a separate RT PCR reaction with the use of the provided PCR control PCRC or Isolation Control lsoC respectively This kit is designed to allow for the testing of 24 samples Kit Components Component Contents Lysis Solution 25 mL Wash Solution 11 mL Elution Buffer 2mL Mini Spin Columns 24 Collection Tubes 24 Elution tubes 1 7 mL 24 Nuclease Free Water 1 25 mL Norgen s DNA Marker 0 1 mL Product Insert 1 IsoC Isolation Control PosC Positive Control The isolation control is a RNA transcript product The positive control is Pospiviroid RNA transcript Customer Supplied Reagents and Equipment e Benchtop microcentrifuge e 1 5 mL microcentrifuge tubes e 96 100 ethanol e Mortar and pestle or other homogenization device Storage Conditions and Product Stability e The Positive Control Pospi PosC red cap and Isolation Control IsoC orange cap sh
15. ould be stored at 70 C If needed make aliquots of the controls according to the volume used in the protocol 10 uL of Pospi PosC or 10 uL of IsoC prior to freezing e The Pospi 2X RT PCR Mastermix green cap and the Control 2X RT PCR Mastermix yellow cap should be stored at 20 C upon receipt 70 C for long term Make appropriate aliquots and store at 20 C if needed e All other kit components may be stored at room temperature e The Pospi 2X RT PCR Mastermix Control 2X RT PCR Mastermix Positive Control and Isolation Control should not undergo repeated freeze thaw a maximum freeze thaw of three times e For RT PCR e Allow reagents to thaw at room temperature prior to use e When thawed mix the components and centrifuge briefly e Work quickly on ice e After addition of RT PCR Mastermix use within one hour General Precautions The user should exercise the following precautions when using the kit e Use sterile pipette tips with filters e Store and extract positive material specimens controls and amplicons separately from all other reagents and add it to the reaction mix in a spatially separated facility e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Work quickly on ice Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Pospiviroid RT PCR Detection Kit i
16. setup of the PCR assay reaction 3 How should it be interpreted if only the Pospiviroid RT PCR control showed amplification but neither the Pospiviroid target nor the Pospiviroid Isolation control amplified for a sample e This indicates a poor isolation The isolation procedure must be repeated 4 How should it be interpreted if only the Isolation Control soC was amplified in a sample e The sample tested can be considered as Pospiviroid negative 5 How should it be interpreted if the Pospiviroid RT PCR control and the Pospiviroid target showed amplification in a sample e The sample tested can be considered positive It could happen when too much template was added to the reaction 6 How should it be interpreted if only the Pospiviroid target and the Pospiviroid PCR control were amplified in a sample e The sample tested can be considered as Pospiviroid positive 7 How should it be interpreted if only the Pospiviroid target was amplified in a sample e The sample tested should be considered as Pospiviroid positive At high Pospiviroid input the Pospiviroid amplicon will be predominant and thus the Pospiviroid PCR control as well as the Pospiviroid Isolation control may not amplify as they compete for PCR resources 8 How should it be interpreted if only the Pospiviroid RT PCR control and the Isolation control showed amplification in a sample e The sample tested can be considered negative 9 What if I forgot to do a dry spin aft
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