Two Great Offers! SPECIAL PROMOTION. 1 FREE Bioreagents with equipment purchase 2 FREE RNA Purification Sample Kit

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1 SPECIAL PROMOTION Two Great Offers! 1 FREE Bioreagents with equipment purchase 2 FREE RNA Purification Sample Kit To get your FREE Fisher BioReagents Purchase any item listed below through Fisher Scientific by 1/31/08. Visit and click on FREE Fisher BioReagents to redeem your free bioreagents. To get your FREE RNA Purification Sample Kit Go to and click on FREE RNA Purification Sample Kit to request your free sample. Cat. No. Description FREE Fisher BioReagents Value Fisher Scientific RNA Purification Kits BP SurePrep TrueTotal RNA Purification Kit $150 BP SurePrep Small RNA Purification Kit $150 Thermo Scientific Microcentrifuges * Sorvall Legend Micro 17 (120V 60 Hz) $ Sorvall Legend Micro 17R (120V 60 Hz) $ Sorvall Legend Micro 21 (120V 60 Hz) $ Sorvall Legend Micro 21R (120V 60 Hz) $300 Thermo Scientific Spectronic BioMate 3 UV/Vis Spectrophotometers V, 50/60Hz $ V, 50/60Hz (built-in printer) $ V, 50/60Hz (6-position automatic cell holder) $ V, 50/60Hz (6-position automatic cell holder/built-in printer) $ BioMate 3 and 0.2mm nanocell bundle $200 Thermo Scientific Upright Revco Freezers Revco Ultima PLUS, 691 Liters (24.2 cu. ft.) $ Revco Ultima PLUS, 572 Liters (20.2 cu. ft.) $ Revco Ultima PlUS, 487 Liters (17.2 cu. ft.) $ Revco Elite PLUS, 691 Liters (24.2 cu. ft.) $ Revco Elite PLUS, 572 Liters (20.2 cu. ft.) $ Revco Value PLUS, 691 Liters (24.4 cu. ft.) $300 *All catalog numbers include 24-place microtube rotor with ClickSeal bio-containment lid. Offer expires on 1/31/2008. Free Fisher BioReagents must be redeemed by 2/28/2008. Valid in the U.S. only. Not valid with any other offers, contracts or agreements. Void where prohibited. Free Fisher BioReagents value based on US list prices. FREE RNA Purification Sample Kit! Visit for details Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. BN BRO-SPSBIOE2-0807

2 BIOESSENTIALS Your guide to vital reagents, consumables and equipment for life science applications Vo l u m e 2 CELL CULTURE Extraction & Purification of Biomolecules PCR & RT-PCR SEPARATION OF NUCLEIC ACIDS BY AGAROSE GEL ELECTROPHORESIS PROTEIN ELECTROPHORESIS FREE RNA Purification Sample Kit! See details on back cover

3 Chance favors the prepared mind. Louis Pasteur Choosing the best reagents and equipment for your protocol will give your RNA research a better chance to succeed by eliminating variables that may alter the results of your molecular biology experiments. As RNA analysis gains the spotlight for assessing the functional characterization of disease-relevant genes for drug discovery and basic research, there is a growing need for more precise and reliable tools to drive successful experimentation in the researcher s lab. In response to evolving needs, Fisher BioReagents introduces the SurePrep RNA purification kits that enable a scientist to capture all sizes of RNA molecules from the biological samples of interest. The SurePrep technology provides purified, full-length large RNAs as well as small RNAs such as microrna (mirna) and short interfering RNA (sirna) that are being investigated as key components in regulating gene expression in signaling pathways, cell death, organ development, metabolism, and disease states. microcentrifuge equipped with high-quality microcentrifuge tubes. The powerful and versatile Thermo Scientific Sorvall Legend Micro 17 and 21 Series is ideally suited for use with the Fisher SurePrep spin columns, Fisher Scientific microcentrifuge tubes. Our microcentrifuge tubes are free of RNase, DNase, and RNA to ensure precise, consistent results. The quality of our Sorvall microcentrifuges and the novel purification technology utilized in SurePrep RNA purification kits combined with reliable consumables will eliminate unwanted variables from your research and increase the likelihood of obtaining information that might otherwise be lost with purification techniques that do not isolate all sizes of intact RNA. For the best results with demanding separations such as RNA purification, it is important to choose a high-performing 1

4 BIOESSENTIALS 4-STEP Selection Process Extraction & Purification of Biomolecules Use the simple 4-step selection process outlined in this guide to choose the appropriate Fisher BioReagents kits, Fisher Scientific consumables, and Thermo Scientific instruments for your biomolecular isolation and purification protocols. 1 Choose Your Purification Protocol Spin Column Chromatography Conventional Guanidinium Method 2 Choose Your Consumables RNase-free tubes 3 Choose Your Equipment Centrifuges for separating RNA Spectrophotometers for Quantifying RNA Freezers for Storing RNA 4 Choose Your Downstream Application Analysis of Gene Expression Gel Electrophoresis Northern Blot Hybridization Ribonuclease Protection Assay (RPA) 2

5 1 PURIFICATION: SPIN COLUMN & CONVENTIONAL Choose your method Isolating clean, intact RNA is essential for the successful analysis of gene expression by Northern hybridization, RPA, or RT-PCR. The RNA must be free of DNA and other cellular macromolecules such as polysaccharides and proteins that can interfere with labeling and hybridization. Fisher BioReagents offers a variety of RNA isolation products specialized for different sample types and for specific downstream applications. Spin Column Chromatography The new SurePrep family of RNA purification kits utilizes spin column chromatography with a proprietary resin that separates RNA from other cellular macromolecules without the use of phenol or chloroform. SurePrep RNA purification kits are suitable for a wide variety of sample types, including plant and animal tissues, cultured cells, fungi, blood, bacteria, and yeast. The SurePrep protocol includes the three essential steps for the successful isolation of intact RNA: 1. Effective disruption of cells or tissue 2. Inactivation of endogenous RNase activity 3. Removal of contaminating DNA and proteins A microcentrifuge equipped with a rotor for 2 ml tubes, such as the Thermo Scientific Sorvall Legend Micro 21R, is required for all centrifugation steps. SurePrep RNA Highlights: Simple, 30-minute procedure Utilizes proprietary resin in spin column format No organic extraction or alcohol precipitation Optimized kits for specific applications RNA suitable for reverse transcription PCR, Northern blotting, RNase protection, primer extension, and expression array analysis TrueTotal RNA Purification Kit With the TrueTotal procedure, all sizes of RNA are purified, including large mrna and ribosomal RNA as well as small RNA species (< 200 nucleotides) such as transfer RNA and micro RNA (mirna). Biological samples are first disrupted in a highly denaturing guanidine thiocyanate solution that simultaneously lyses cells and inactivates endogenous ribonucleases to ensure purification of intact RNA. The lysate is then diluted with ethanol to provide the appropriate binding conditions. The sample is then applied to a SurePrep spin column where the total RNA binds to the resin and most other cellular contaminants are effectively washed away. Up to 50 µg of high-quality total RNA is then eluted in 50 µl of low ionic strength buffer. 3

6 PURFICATION 4000 nt SurePrep Competitor M nt 1000 nt 23S 16S 500 nt Table 1: Maximum Amount of Starting Material Material Animal Cells Animal Tissues Blood Bacteria Yeast Fungi Plant Tissues Average Yields* HeLa Cells (1 x 10 6 cells) Amount 3 x 10 6 cells 25 mg 100 ul 1 x 10 9 cells 1 x 10 8 cells 50 mg 50 mg 15 ug RNA E. coli (1 x 10 9 cells) 50 ug RNA *Average yields will vary depending on a number of factors including species, growth conditions, and developmental stage. 200 nt 25 nt small RNA Resolution of E. coli Total RNA by Capillary Electrophoresis 1 µl of purified sample RNA loaded onto an Agilent RNA 6000 Nano Chip and resolved using Agilent 2100 Bioanalyzer. Note: the small molecular weight RNA isolated by the new SurePrep family of RNA purification kits (lanes 1 & 2) versus competitor s kit (lanes 3 & 4). MW standard in lane 1. SurePrep Small RNA Purification Kit Small regulatory RNA molecules play an important role in regulating gene expression by binding to the target mrnas. The conventional protocols for isolating total RNA and mrna are not optimized for isolation of small RNA, resulting in the loss of significant amounts of small RNA. Moreover, co-purification of larger RNA molecules with small RNA inhibits expression analysis of small RNA. The SurePrep Small RNA Purification Kit provides a rapid and efficient method to isolate and purify small RNA molecules (< 200 nt) from larger RNAs in various biological samples. These small RNAs include regulatory RNA molecules, such as micro RNA (mirna) and short interfering RNA (sirna), as well as trna and 5s rrna. Large RNA molecules such as 28S rrna and mrna are efficiently removed in this kit using the Large RNA Removal Column, whereas small RNAs are isolated in the Small RNA Enrichment Column (see Table 2 on page 5). Purified small RNA from the SurePrep kit is suitable for most downstream applications related to gene regulation and functional analysis. See our RNA Purification Troubleshooting Guide on page 10 4

7 PURIFICATION: SPIN COLUMN & CONVENTIONAL (continued) Table 2: SurePrep RNA Kit Selection Guide Cat. No. Description Number of preps per kit Recommended applications BP TrueTotal RNA Purification Kit 50 Total RNA isolation including mirna and sirna from varity of samples BP Small RNA Purification Kit 25 Enrichment of small RNA by removing large RNA species for studying gene expression level and function of mirna and sirna BP RNA/DNA/Protein Purification Kit 50 Separation and purification of total RNA, genomic DNA, and protein from single sample in < 20min using same column BP Urine Exfoliated Cell RNA Purification Kit 50 Isolation and purification of total RNA from urinary tract exfoliated cells obtained from urine sample. BP Urine Bacterial RNA Purification Kit 50 Bacterial total RNA isolation and purification from either human urine samples or from the urine samples of animals. Isolates RNA from both gram positive and gram negative bacteria BP Nuclear or Cytoplasmic RNA Purification Kit 50 (Cyto) 25/25 (Nuc & Cyto) Isolate and purify both cytoplasmic and nuclear RNA from cultured cells or animal tissue. Allows to enrich for cytoplasmic or nuclear RNA depending on the downstream application BP RNA/Protein Purification Kit 50 Isolate and purify total RNA and proteins simultaneously from a single sample using the same column in <20min BP Leukocyte RNA Purification Kit 50 Isolation and purification of white blood cells total RNA from mamalian blood samples BP RNA Cleanup and Concentration Kit 50 Purifies, cleans and concentrates RNA isolated using other methods such as phenol:chloroform extraction or after enzymatic reactions Conventional Guanidinium Method Many investigators still use the conventional, single-step technique of Chomczynski and Sacchi (1987) to isolate total RNA from eukaryotic cells. In this method, cells are lysed using guanidinium thiocyanate and the RNA extracted from the homogenate with phenol:chloroform at a reduced ph. The total RNA is of high quality, and the yield is about 5 µg/mg of starting tissue. Many of the reagent chemicals used in this traditional RNA purification protocol are available through Fisher BioReagents. Table 3: Fisher BioReagents products for Conventional RNA Purification Protocol Cat. No. BP2438 BP221 BP327 BP176 BP333 Description Phosphate Buffered Saline, 1X Solution Guanidine Thiocyanate Sodium Citrate Dihydrate 2-Mercaptoethanol Sodium Acetate Anhydrous BP1751l Phenol, Saturated ph 4.3 BP1145 BP1150 Chloroform Isoamyl Alcohol 5

8 2 CONSUMABLES: RNASE-FREE MICROCENTRIFUGE TUBES Choose your consumables To ensure accurate, repeatable results for your RNA purification protocols, our Fisher Scientific microcentrifuge tubes are produced in an ISO 9001 manufacturing facility, which guarantees that the non-sterile product is delivered to you free of RNase, DNase and DNA. Our sterile, individually wrapped tubes offer additional product cleanliness by certifying they are also free of pyrogens, endotoxins, bioburden, and PCR inhibitors. All microcentrifuge tubes can be boiled, autoclaved up to 121 C, 15 PSI for 10 minutes and are freezable down to -80 C. Highlights: Precision molded polypropylene with leak-proof snap-seal closure allows repeated openings and closings, yet resists inadvertent opening from internal pressure while centrifuged or boiled An easy-open, flexible cap collar design not only resists but also contains splashing The crystal-clear design allows for an excellent view of the contents A pierceable cap with a smooth-walled conical bottom provides visibility for you to guide the tip for maximum sample retrieval Variety of colors makes sample tracking and monitoring easy Available in 0.6ml, 1.5ml and 2.0ml sizes Choose from tubes that are plain without any markings, frosted cap and side, and with graduations Table 4: Fisher Scientific Microcentrifuge Tube Selection Guide Cat. No. Description ml graduated non-sterile microcentrifuge tube, frosted cap and side-writing pad ml graduated sterile microcentrifuge tube, frosted cap and side-writing pad ml graduated non-sterile microcentrifuge tube, frosted cap and side-writing pad ml graduated sterile microcentrifuge tube, frosted cap and side-writing pad ml capless graduated non sterile microcentrifue tubes ml capless graduated non sterile microcentrifue tubes Tubes Bags Per Graduation Tubes Per Bag Case g-force Markings Available ,000 x g 0.1, 0.5, 1.0, 1.5 ml colors 250* 10 26,000 x g 0.1, 0.5, 1.0, 1.5 ml clear only ,000 x g 0.5, 1.0, 1.5, 2.0 ml colors 250* 10 25,000 x g 0.5, 1.0, 1.5, 2.0 ml clear only ,000 x g 0.1, 0.5, 1.0, 1.5 ml clear only ,000 x g 0.5, 1.0, 1.5, 2.0 ml clear only 6

9 3 EQUIPMENT: CENTRIFUGES, SPECTROPHOTOMETER, FREEZERS Choose your equipment Centrifuges for Separating RNA Ideally suited to RNA applications, the Thermo Scientific Sorvall Legend Micro 17 & 21 Series is available in two g-force ranges 17,000 and 21,000 x g and in refrigerated and non-refrigerated models. The 24-place rotor is uniquely designed to accommodate the Fisher Scientific SurePrep spin columns. Fast acceleration and deceleration rates on all models help you process samples quickly, and a wide selection of rotors gives you added flexibility for other microcentrifuge protocols. Highlights Choice of 17,000 x g and 21,100 x g in both ventilated and refrigerated models Very quiet refrigerated: 50 db(a); ventilated: 56 db(a) ClickSeal bio-containment lid provides superior safety and convenience 24 x 1.5 / 2ml rotor with ClickSeal bio-containment lidcomes standard Practical lid design allows rotor to accommodate SurePrep spin columns Fast acceleration/deceleration: 12 sec Five rotors to choose from, including a unique dual-row rotor that spins 18 each of 1.5/2.0 ml and 0.5 ml tubes at the same time eliminating the need for adapters The large LED display and intuitive controls make operation easy; a pulse capability allows you to toggle between RPM and RCF Table 5: Thermo Scientific Sorvall Legend Microcentrifuge Ordering Information Cat. No. Description* Max RPM / RCF Ventilated Sorvall Legend Micro V 60 Hz Sorvall Legend Micro V 60 Hz Refrigerated Sorvall Legend Micro 17R 120V 60 Hz Sorvall Legend Micro 21R 120V 60 Hz 13,300 / 17,000 14,800 / 21,100 13,300 / 17,000 14,800 / 21,100 * All catalog numbers include 24-place microtube rotor with ClickSeal bio-containment lid. 7 For more information on our full range of centrifuge solutions, visit

10 EQUIPMENT Spectrophotometers for Quantifying RNA The concentration and purity of an RNA solution can be determined by measuring its absorbance at 260 and 280 nm using the Thermo Scientific Spectronic BioMate 3 UV-VIS Spectrophotometer. This compact, easy-to-use instrument provides pre-programmed procedures to quickly measure concentrations of dsdna, ssdna, RNA, and oligonucleotides. To determine the RNA concentration in µg/ml, multiply the A260 by the dilution factor and the extinction coefficient. A260 x dilution factor x 40 = µg RNA/ml The ratio of A260 to A280 is a measure of RNA purity, with a value >1.8 indicating the RNA sample is reasonably clean of proteins that could interfere with downstream applications. Table 6: Thermo Scientific Spectronic BioMate 3 UV/Vis Spectrophotometer Ordering Information Cat. No. Description V, 50/60Hz, US line cord V, 50/60Hz, US line cord/built-in printer V, 50/60Hz, US line cord 6-position automatic cell holder V, 50/60Hz, US line cord 6-position automatic cell holder/built-in printer BioMate 3 and 0.2mm nanocell bundle Spectronic BioMate 3 Freezers for Storing RNA Purified RNA in elution buffer from SurePrep kits may be stored at -20 C for a few days, but for long term storage -70 C is recommended. Thermo Scientific Revco PLUS -86 C Freezers deliver the best protection of critical nucleic acid samples using advanced refrigeration technology. Samples are safeguarded by combining the highest temperature stability and fastest temperature recovery. Table 7: Thermo Scientific Freezer Selection Guide Volume Cat. No. Description Liters (cu. ft.) Revco Ultima PLUS 691 (24.2) Revco Ultima PLUS 572 (20.2) Revco Ultima PlUS 487 (17.2) Revco Elite PLUS 691 (24.2) Revco Elite PLUS 572 (20.2) Revco Value PLUS 691 (24.4) For more information on our full range of cold storage solutions, visit 8

11 4 DOWNSTREAM APPLICATIONS FOR GENE EXPRESSION ANALYSIS Choose your downstream application One of the primary uses of RNA isolation procedures is the analysis of gene expression. RNA purified by SurePrep kits is suitable for a number of techniques that will assess the abundance of a particular RNA species or the structural detail of an RNA transcript. Gel Electrophoresis After RNA is extracted from the sample, it is usually first separated by electrophoresis on a denaturing agarose gel to verify its integrity or to separate various RNA species for further analysis. This separation can be carried out with a submarine electrophoresis gel box such as the Thermo Scientific Owl Easycast B1 gel box and low voltage power supplies. In addition, Fisher BioReagents offers functionally-tested reagents and buffers for agarose gel electrophoresis. Northern Blot Hybridization Northern blot analysis is used to determine the size and amount of a particular RNA species. The hybridization process is carried out in a temperature-controlled chamber such as the Thermo Scientific Hybaid Shake & Stack. This oven provides unmatched reliability, accuracy and uniformity to ensure reproducible results. Fisher BioReagents offers functionally tested reagents and buffers for the hybridization process. Ribonuclease Protection Assay (RPA) RNase protection assays are used to measure the abundance of specific mrnas. RPA is superior to Northern blots for detecting and quantifying low-abundance RNAs. In the RPA process, the RNA target and an RNA probe are hybridized in solution. The hybridization mixture is placed in a temperature-controlled water bath set at the annealing temperature. Our Thermo Scientific Precision General Purpose Water Baths provide exceptional performance and reliability for the RPA hybridization process. Thermo Scientific Owl Easycast B1 Gel Box Thermo Scientific Hybaid Shake & Stack Hybridization Oven Thermo Scientific Precision General Purpose Water Baths 9 For more information on the full range of Thermo Scientific laboratory equipment solutions, visit

12 SurePrep RNA Purification Troubleshooting Guide Problem Possible Cause Solution and Explanation Incomplete lysis of cells or tissue Ensure that the appropriate amount of Lysis Solution was used for the amount of cells or tissue. Poor RNA recovery Clogged column RNA is degraded RNA does not perform well in downstream applications Genomic DNA contamination Column has become clogged An alternative elution solution was used Ethanol was not added to the lysate Ethanol was not added to the Wash Solution Low RNA content in cells or tissues used Cell culture: Cell monolayer not washed with PBS Yeast: Lyticase was not added resuspension buffer Bacteria and yeast: All traces media not removed Insufficient solubilization of cells or tissues Maximum number of cells or amount of tissue exceeds kit specifications Clarified lysate was not used for the binding step High amounts of genomic DNA present in sample Centrifuge temperature too low RNase contamination Cell lysis procedure not performed quickly enough Improper storage of the purified RNA Frozen tissues or cell pellets were allowed to thaw prior to RNA isolation Starting material may have high RNase content Lysozyme or lyticase used may not be RNase-free RNA was not washed 3 times with the provided Wash Solution Ethanol carryover Large amounts of starting material used Do not exceed the recommended amounts of starting biological material. The amount of starting material may need to be decreased if the column shows clogging below the recommended levels. See also Clogged Column below. It is recommended that the Elution Buffer supplied with this kit be used for maximum RNA recovery. Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column. Ensure that 50 ml of 95% ethanol is added to the supplied Wash Solution prior to use. Different tissues and cells have different RNA contents, and thus the expected yield of RNA will vary greatly from these different sources. Please check literature to determine the expected RNA content of your starting material. Ensure that the cell monolayer is washed with the appropriate amount of PBS in was order to remove residual media from cells. Ensure that the appropriate amount of lyticase is added when making the to the Resuspension Buffer. Ensure that all media is removed prior to the addition of the lysis solution through of aspiration. Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue. Refer to specifications to determine if amount of starting material falls within kit specifications Ensure that after the lysis step, the sample is centrifuged if any precipitates are present, and that only the clarified lysate is used in subsequent steps. The lysate may be passed through a 25 gauge needle attached to a syringe 5-10 times in order to shear the genomic DNA prior to loading onto the column. Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 20 C may cause precipitates to form that can cause the columns to clog. RNases may be introduced during the use of the kit. Ensure proper procedures are followed when working with RNA. Please refer to Working with RNA in Section I of this user guide. In order to maintain the integrity of the RNA, it is important that the procedure be performed quickly. This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol, since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized. For short term storage RNA samples may be stored at -20 C for a few days. It is recommended that samples be stored at -70 C for longer term storage. Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised. For starting materials with high RNase content, it is recommended that 2-mercaptoethanol be added to the Lysis Solution. Ensure that the lysozyme and lyticase being used with this kit is RNase-free, in order to prevent possible problems with RNA degradation. Traces of salt from the binding step may remain in the sample if the column is not washed 3 times with Wash Solution. Salt may interfere with downstream applications, and thus must be washed from the column. Ensure that the dry spin under the Column Wash procedure is performed, in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications. Perform RNase-free DNaseI digestion on the RNA sample after elution to remove genomic DNA contamination. 10 RNA purification Troubleshooting Guide