RNA Control 250 User Guide
Contents
1. After thawing for exactly 5 min remove the tube of RNA Control 250 from the water bath and vortex vigorously for 10 seconds Centrifuge the tube of RNA Control 250 for 30 seconds at 2 300 x g 5 000 rpm in a microcentrifuge to collect the liquid at the bottom of the tube Store the RNA Control 250 at room temperature while you proceed immediately to step 2 2 Prepare the NanoDrop Spectrophotometer for spectral measurements a Raise the sampling arm of the spectrophotometer and clean the lower and upper sample pedestals by rinsing with nuclease free water as recommended by the manufacturer Wipe off any excess water with a lint free disposable tissue Launch the NanoDrop Spectrophotometer program to initiate the measurement software as described in the User s Manual The main application window will appear Raise the sampling arm and using a calibrated P 2 or P 10 pipette transfer 2 0 uL of nuclease free water to the lower sample pedestal Click OK to initialize the spectrophotometer After initialization select RNA 40 as the sample type Raise the sample arm and dry the water from both the lower and upper sample pedestals with a lint free disposable tissue Transfer 2 0 uL of THE RNA Storage Solution to the lower sample pedestal then lower the sampling arm Blank the spectrophotometer Raise the sampling arm and wipe off any buffer from the top and bottom pedestals with a lint2. handling and disposal limitations may apply Documentation and Support Obtaining SDSs Documentation and Support Obtaining SDSs Safety Data Sheets SDSs are available from www lifetechnologies com sds Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtaining support For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches 10 RNA Control 250 User Guide Documentation and Support Obtaining support RNA Control 250 User Guide 11 Ul O Hsst For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Limited Use Label License Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its componen
3. Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials e Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en RNA Control 250 User Guide Chemical safety RNA Control 250 User Guide Troubleshooting Chemical safety A WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions
4. Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special
5. m bioanalyzer 2 a 250 m bioanalyzer 3 2 200 m NanoDrop 150 F 100 5 l _ Ei TI 1 2 1 4 1 8 Dilution 6 RNA Control 250 User Guide Confirm well to well reproducibility using the Agilent 2100 Bioanalyzer instrument Troubleshooting Confirm well to well reproducibility using the Agilent 2100 Bioanalyzer instrument The RNA Control 250 is also ideal for validating well to well reproducibility and dilution linearity on the Agilent 2100 Bioanalyzer instrument Figure 3 on page 6 Triplicate samples of a dilution series of RNA Control 250 were analyzed on three different Agilent 2100 Bioanalyzer instruments Panel A shows the gel like images from one Bioanalyzer instrument along with the indicated benchmark concentrations determined by NanoDrop measurements Panel B shows the mean concentrations derived from area under curve AUC measurements on each Bioanalyzer instrument compared to the benchmark values Troubleshooting Observation Possible cause Recommended action RNA concentration measured on the NanoDrop spectrophotometer is outside specifications Air bubbles formed in the RNA solution during placement of the sample on the lower pedestal Remove air bubbles and repeat the assay RNA was not properly thawed and resuspended Carefully repeat thaw procedure at 37 C using a water bath to thaw the RNA step 1 on page 4 Pipettor P 10 is not properly calibrated assa
6. using RNA Control 250 Generate accurate RNA concentration standard curves Figure 2 RNA Control 250 concentration curve A standard generated using RNA Control 250 and measured using RiboGreen Reagent Molecular Probes Cat R11491 on a NanoDrop ND 3300 Fluorospectrometer 1800 1600 1400 1200 1000 800 600 400 200 0 R 0 997 Fluorescence 0 15 30 45 60 75 ng per reaction Figure 3 RNA Control 250 on the Agilent 2100 Bioanalyzer instrument Triplicate samples of a dilution series of RNA Control 250 were analyzed on three different Agilent 2100 Bioanalyzer instruments using an RNA LabChip Kit Panel A Gel like images from one Bioanalyzer instrument Indicated concentrations were calculated from NanoDrop measurements The Ambion RNA 6000 Ladder Part no AM7152 is shown at left Panel B Mean concentrations derived from area under curve AUC measurements of triplicate samples on each Bioanalyzer instrument For comparison the benchmark concentration values based on NanoDrop measurements are shown The AUC measurements decrease linearly with dilution however Life Technologies routinely observes that RNA concentrations derived from Bioanalyzer instrument AUC measurements are higher than benchmark concentrations determined by NanoDrop measurements es ee ee ee ee a 2002 nt ng L 250 125 62 5 31 25 450 400 350 o bioanalyzer 1 300
7. A concentration standard curves RNA Control 250 User Guide RNA Control 250 was designed for verifying performance of NanoDrop Spectrophotometers with the sample type most commonly measured on the instrument This highly defined and precisely controlled RNA product can ensure the user of run to run accuracy and measurement reproducibility when used on NanoDrop Spectrophotometers following the procedures in this protocol To illustrate this triplicate aliquots of RNA Control 250 were measured on 12 different NanoDrop spectrophotometers Figure 1 Measurements fell within 5 of the mean concentration value for this lot of RNA Control 250 The RNA Control 250 makes an ideal reference standard for generating concentration standard curves using RiboGreen reagent such as shown in Figure 2 on page 6 Figure 1 Reproducibility of RNA Control 250 measurements Bars represent the average of triplicate samples of RNA Control 250 measured on 12 different NanoDrop spectrophotometers The mean concentration for this lot of RNA Control 250 was 256 ng uL 280 270 260 250 240 230 220 210 200 1 2 3 4 5 6 7 8 9 10 1 12 Instrument Number Note that the accuracy and linearity of any RNA concentration standard curve depends not only on the accuracy of the RNA reference standard used for making the serial dilutions but also on the accuracy and precision of the pipette used to make the dilutions Applications
8. USER GUIDE ambion by Lefe technologies RNA Control 250 Publication Part Number 7155M Rev C Revision Date September 2011 E Product description 6 eee eeeeee ene 1 M Product SpecificatiOn ocs 0 ii tero da tg sano se a Mods WS eases os HSN 2 m Kit contents and storage 6 nents 2 Mm Required materials ss sis ccehe scents cree eeepc ee eae ees bee ee edrr sarri 3 m RNA Control 250 User Procedure 1 6 6 6 3 m Applications using RNA Control 250 2 0 0 6 ene 5 M Troubleshooting 6 kiiin es ence eee eee e teen een ees 7 Re gga a E EEEE ERRA EREE EEE 8 m Documentation and Support 6 eee eens 10 Product description The RNA Control 250 provides a precise accurate and reproducible RNA concentration reference for use in conjunction with NanoDrop Spectrophotometers The control consists of a single ultra pure 2 002 nt RNA transcript of precisely defined sequence molecular weight and concentration It is packaged in special non stick tubes in THE RNA Storage Solution Part no AM7000 AM7001 The RNA Control 250 also makes an ideal reference standard for generating RNA concentration standard curves e g for RiboGreen based assays as well as for validating well to well concentration accuracy and dilution linearity on the Agilent 2100 Bioanalyzer instrument To ensure maximum utility of the RNA Control 250 as a reference tool we recommend that you closely follow the notes in the se
9. ction Minimize user controlled variability on page 3 technologies Product specification Special handling precautions Product specification Parameter Specification Concentration See accompanying specification sheet Storage Buffer THE RNA Storage Solutiont A260 A280 See accompanying specification sheet A260 A230 See accompanying specification sheet A240 See accompanying specification sheet Molecular weight 641 991 Daltons Length 2 002 nt Nucleotide composition A 452 G 499 C 576 U 475 Extinction coefficient E260 40 uL ng cm t 1 mM sodium citrate pH 6 4 0 2 Kit contents and storage Special handling precautions The kit contains sufficient reagents for 10 measurements of RNA Control 250 5 measurements from each of the supplied tubes Product Amount Storage RNA Control 250 2 x 20 uL 20 C THE RNA Storage Solution 100 uL 20 C For short term storage store both tubes of RNA Control 250 at 20 C Do not store in a frost free freezer For long term storage gt 6 months store at 80 C e Do not expose each tube of the RNA Control 250 to more than 5 freeze thaw cycles e Do not sample from any given tube more than 5 times e Always use a new pipette tip when sampling from the tube of RNA Control 250 e Do not dilute the RNA Control 250 when measuring with a NanoDrop Spectrophotometer RNA Control 250 Us
10. er Guide Required materials Minimize user controlled variability Required materials NanoDrop Spectrophotometer Lint free disposable tissue for cleaning the spectrophotometer pedestal Calibrated low volume pipettor e g P 2 or P 10 RNase free Tips e g Part no AM12635 AM12640 Nuclease free Water for cleaning the spectrophotometer e g Part no AM9930 Water bath set to 37 C Optional Reagents and equipment for any other methodology for which you are using the RNA Control 250 as a benchmark e g RiboGreen reagent Molecular Probes NanoDrop Fluorospectrometer or Agilent Bioanalyzer instrument RNA Control 250 User Procedure Note These instructions assume a working knowledge of NanoDrop Spectrophotometers Refer to the NanoDrop User s Manual available at www nanodrop com for detailed instructions on the instrument s use Minimize user User controlled factors can adversely affect both measurement accuracy and controlled reproducibility when using the RNA Control 250 To achieve maximally reproducible variability and accurate concentration readings with the RNA Control 250 5 use these best RNA Control 250 User Guide practices Do not expose each tube of RNA Control 250 to more than 5 freeze thaw cycles Do not dilute the RNA Control 250 Do not sample from the same tube of RNA Control 250 more than 5 times sampling the RNA Control 250 tube too many times can result in excess sample evapo
11. fety AN AN WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document e Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device e Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices
12. free disposable tissue Transfer a new 2 0 uL aliquot of THE RNA Storage Solution to the lower sample pedestal lower the sampling arm and measure the solution The baseline spectrum should be flat and the A26 should indicate an RNA concentration less than 2 ng uL If not repeat steps f h until this is achieved 3 Measure the concentration of the RNA Control 250 in duplicate a b Thoroughly mix the thawed RNA Control 250 by pipetting up and down several times immediately before sampling Transfer 2 0 uL of the RNA Control 250 to the lower sample pedestal with a clean pipette tip RNA Control 250 User Guide Applications using RNA Control 250 Verify performance of NanoDrop Spectrophotometer c Quickly lower the sampling arm and immediately measure the concentration of the sample Note After the RNA Control 250 is placed on the instrument measure the sample quickly within 10 seconds to offset potential sample evaporation Excess evaporation can lead to reduced measurement accuracy d Record the sample concentration The expected value is listed on the specification sheet accompanying the product e Raise the sampling arm and wipe off any solution from the top and bottom pedestals with a lint free disposable tissue f Measure a second aliquot of RNA Control 250 using the same procedure Applications using RNA Control 250 Verify performance of NanoDrop Spectrophotometer Generate accurate RN
13. ration each tube supplies an excess of RNA Control 250 to minimize the effects of evaporation during sampling Thaw mix and handle the RNA Control 250 according to the protocol to maintain its integrity and concentration Use a properly calibrated pipette Always measure the RNA Control 250 using a 2 uL assay volume Although NanoDrop Spectrophotometer measurements are generally volume independent for samples of 1 2 uL in our experience accuracy and reproducibility are greatest when using a 2 uL aliquot Measure the RNA concentration on the NanoDrop Spectrophotometer sample pedestal immediately after dispensing within 10 seconds If measurement is delayed sample evaporation after loading the sample pedestal can reduce accuracy Measure duplicate samples to give the most accurate measurement of the RNA Control 250 Use a properly calibrated spectrophotometer RNA Control 250 User Procedure Begin the procedure Begin the 1 Thaw the RNA Control 250 at 37 C for 5 min procedure a Prior to use remove the RNA Control 250 from the 20 C freezer and thaw the RNA solution by incubating the tube at 37 C in a water bath for exactly 5 min set a timer IMPORTANT Thawing times gt 5 min can lead to decreased transcript integrity over time We recommend using a water bath to thaw the RNA Control 250 because its packaging tube requires a water bath s efficient heat transfer to thaw the RNA Control 250 within 5 min
14. ts is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 2011 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Bioanalyzer is a trademark of Agilent Technologies Inc NanoDrop is a registered trademark of NanoDrop Technologies LLC Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA d Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www appliedbiosystems com support technologies www lifetechnologies com
15. y volumes lt 1 5 uL can increase measurement variability Use a properly calibrated pipettor to make sure that a 2 uL sample is evaluated in the assay Sample pedestal was not properly rinsed and cleaned before use Clean the sample pedestal with nuclease free water and repeat the assay Spectrophotometer was not properly blanked Reblank the spectrophotometer as described in step 2 on page 4 Spectrophotometer is outside normal calibration parameters Recalibrate the spectrophotometer according to manufacturer s recommendations A240 A280 or A260 A230 ratios are outside specifications Dust or other particles on sample pedestal cause light scattering Clean sample pedestal using nuclease free water and lint free disposable tissues Spectrophotometer is not accurately calibrated Recalibrate spectrophotometer according to the manufacturer s recommendations RNA Control 250 is degraded RNA Control 250 has been thawed under elevated temperatures and or for extended periods of time Minimize exposure to elevated temperatures and prolonged thawing RNA Control 250 has been contaminated with exogenous ribonucleases Use gloves and RNase free tips Never reuse RNA Control 250 after it has been placed on the pedestal because the pedestal is not RNase free RNA Control 250 User Guide Troubleshooting Biological hazard safety Appendix A Safety Biological hazard sa
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