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Cat. No. DescriptionFREE Fisher

BioReagents Value

Fisher Scientific RNA Purification Kits

BP2800-50 SurePrep™ TrueTotal™ RNA Purification Kit $150

BP2801-25 SurePrep™ Small RNA Purification Kit $150

Thermo Scientific Microcentrifuges *

75002431 Sorvall Legend Micro 17 (120V 60 Hz) $300

75002441 Sorvall Legend Micro 17R (120V 60 Hz) $300

75002436 Sorvall Legend Micro 21 (120V 60 Hz) $300

75002446 Sorvall Legend Micro 21R (120V 60 Hz) $300

Thermo Scientific Spectronic BioMate 3 UV/Vis Spectrophotometers

14-298-2081 100-240V, 50/60Hz $200

14-298-2082 100-240V, 50/60Hz (built-in printer) $200

14-298-2073 100-240V, 50/60Hz (6-position automatic cell holder) $200

14-298-2074 100-240V, 50/60Hz (6-position automatic cell holder/built-in printer) $200

14380505 BioMate 3 and 0.2mm nanoCell bundle $200

Thermo Scientific Upright Revco Freezers

13-989-721 Revco Ultima PLUS, 691 Liters (24.2 cu. ft.) $300

13-989-719 Revco Ultima PLUS, 572 Liters (20.2 cu. ft.) $300

13-989-717 Revco Ultima PlUS, 487 Liters (17.2 cu. ft.) $300

13-989-715 Revco Elite PLUS, 691 Liters (24.2 cu. ft.) $300

13-989-706 Revco Elite PLUS, 572 Liters (20.2 cu. ft.) $300

13-989-713 Revco Value PLUS, 691 Liters (24.4 cu. ft.) $300

*All catalog numbers include 24-place microtube rotor with ClickSeal bio-containment lid.

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CELL CULTURE

Extraction & Purification of Biomolecules

PCR & RT-PCR

SEPARATION OF NUCLEIC ACIDS BY AGAROSE GEL ELECTROPHORESIS

PROTEINELECTROPHORESIS

BIOESSENTIALS™

Your guide to vital reagents, consumables and equipment for life science applications

Volume 2

FREE RNA Purification Sample Kit! See details on back cover

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.

For the best results with demanding

separations such as RNA purification, it is

important to choose a high-performing

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.

“Chance favors the prepared mind.” — Louis Pasteur

Choosing the best reagents and equipment for your protocol will give yourRNA research a better chance to succeed by eliminating variables that mayalter the results of your molecular biology experiments.

1

BIOESSENTIALS4-STEP Selection Process

2

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)

3

1 PURIFICATION: SPIN COLUMN & CONVENTIONAL

TrueTotal™ RNA Purification KitWith 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 andmicro RNA (miRNA). Biological samples are firstdisrupted in a highly denaturing guanidine thiocyanate solution that simultaneously lysescells and inactivates endogenous ribonucleasesto ensure purification of intact RNA. The lysate is then diluted with ethanol to provide the appropriate binding conditions. The sampleis then applied to a SurePrep spin column wherethe total RNA binds to the resin and most othercellular contaminants are effectively washedaway. Up to 50 µg of high-quality total RNA isthen eluted in 50 µl of low ionic strength buffer.

Spin Column ChromatographyThe new SurePrep family of RNA purification kits utilizes spin column chromatography with aproprietary 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 sampletypes, 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 tissue2. 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 SorvallLegend 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, primerextension, and expression array analysis

Isolating clean, intact RNA is essential for thesuccessful analysis of gene expression byNorthern hybridization, RPA, or RT-PCR. TheRNA must be free of DNA and other cellularmacromolecules such as polysaccharides and

proteins that can interfere with labeling andhybridization. Fisher BioReagents® offers avariety of RNA isolation products specializedfor different sample types and for specificdownstream applications.

Choose your method

4

SurePrep Small RNA Purification KitSmall regulatory RNA molecules play an important role in regulating gene expression by bindingto 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 andpurify small RNA molecules (< 200 nt) from larger RNAs in various biological samples. Thesesmall RNAs include regulatory RNA molecules, such as micro RNA (miRNA) and short interferingRNA (siRNA), as well as tRNA and 5s rRNA. Large RNA molecules such as 28S rRNA and mRNAare efficiently removed in this kit using the Large RNA Removal Column, whereas small RNAsare isolated in the Small RNA Enrichment Column (see Table 2 on page 5). Purified small RNAfrom the SurePrep kit is suitable for most downstream applications related to gene regulationand functional analysis.

Resolution of E. coli Total RNA by Capillary Electrophoresis1 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.*Average yields will vary depending on a number of factors including

species, growth conditions, and developmental stage.

PURFICATION

Table 1: Maximum Amount of Starting Material

See our RNA Purification Troubleshooting Guide on page 10

Material Amount

Animal Cells 3 x 106 cells

Animal Tissues 25 mg

Blood 100 uL

Bacteria 1 x 109 cells

Yeast 1 x 108 cells

Fungi 50 mg

Plant Tissues 50 mg

Average Yields*

HeLa Cells (1 x 106 cells) 15 ug RNA

E. coli (1 x 109 cells) 50 ug RNA

4000 nt —

2000 nt —

1000 nt —

500 nt —

200 nt —

25 nt —

M 1 2 3 4

SurePrep Competitor

23S

16S

small RNA

5

Conventional Guanidinium MethodMany investigators still use the conventional,single-step technique of Chomczynski andSacchi (1987) to isolate total RNA fromeukaryotic cells. In this method, cells arelysed using guanidinium thiocyanate and theRNA extracted from the homogenate withphenol:chloroform at a reduced pH. The totalRNA is of high quality, and the yield is about5 µg/mg of starting tissue. Many of thereagent chemicals used in this traditionalRNA purification protocol are availablethrough Fisher BioReagents.

PURIFICATION: SPIN COLUMN & CONVENTIONAL (continued)

Table 2: SurePrep RNA Kit Selection Guide

Table 3: Fisher BioReagents products forConventional RNA Purification Protocol

Cat. No. Description

Number of prepsper kit Recommended applications

BP2800-50 TrueTotal RNA Purification Kit 50 Total RNA isolation including miRNA and siRNA from varity of samples

BP2801-25 Small RNA Purification Kit 25 Enrichment of small RNA by removing large RNAspecies for studying gene expression level and function of miRNA and siRNA

BP2802-50 RNA/DNA/Protein Purification Kit 50 Separation and purification of total RNA, genomicDNA, and protein from single sample in < 20min usingsame column

BP2803-50 Urine Exfoliated Cell RNA Purification Kit 50 Isolation and purification of total RNA from urinary tract exfoliated cells obtained from urine sample.

BP2804-50 Urine Bacterial RNA Purification Kit 50 Bacterial total RNA isolation and purification fromeither human urine samples or from the urine samples of animals. Isolates RNA from both grampositive and gram negative bacteria

BP2805-50 Nuclear or Cytoplasmic RNA Purification Kit

50 (Cyto)25/25

(Nuc &Cyto)

Isolate and purify both cytoplasmic and nuclear RNAfrom cultured cells or animal tissue. Allows to enrichfor cytoplasmic or nuclear RNA depending on thedownstream application

BP2806-50 RNA/Protein Purification Kit 50 Isolate and purify total RNA and proteins simultaneously from a single sample using the same column in <20min

BP2807-50 Leukocyte RNA Purification Kit 50 Isolation and purification of white blood cells totalRNA from mamalian blood samples

BP2809-50 RNA Cleanup and Concentration Kit 50 Purifies, cleans and concentrates RNA isolated usingother methods such as phenol:chloroform extractionor after enzymatic reactions

Cat. No. Description

BP2438 Phosphate Buffered Saline, 1X Solution

BP221 Guanidine Thiocyanate

BP327 Sodium Citrate Dihydrate

BP176 2-Mercaptoethanol

BP333 Sodium Acetate Anhydrous

BP1751l Phenol, Saturated pH 4.3

BP1145 Chloroform

BP1150 Isoamyl Alcohol

Choose your consumables

Highlights:

• Precision molded polypropylene with leak-proof snap-seal closure allows repeated openingsand 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

6

CONSUMABLES: RNASE-FREE MICROCENTRIFUGE TUBES

To ensure accurate, repeatable results for your RNA purification protocols, ourFisher Scientific microcentrifuge tubes areproduced in an ISO 9001 manufacturing facility, which guarantees that the non-sterileproduct 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.

2

Table 4: Fisher Scientific Microcentrifuge Tube Selection Guide

Cat. No. DescriptionTubes

Per BagBags Per

Case g-forceGraduationMarkings

TubesAvailable

02-682-550 1.5ml graduated non-sterile microcentrifuge tube, frosted cap and side-writing pad

500 10 26,000 x g 0.1, 0.5, 1.0, 1.5 ml colors

02-681-5 1.5ml graduated sterile microcentrifuge tube, frosted cap and side-writing pad

250* 10 26,000 x g 0.1, 0.5, 1.0, 1.5 ml clear only

02-681-258 2.0ml graduated non-sterile microcentrifuge tube, frosted cap and side-writing pad

500 10 25,000 x g 0.5, 1.0, 1.5, 2.0 ml colors

02-681-271 2.0ml graduated sterile microcentrifuge tube, frosted cap and side-writing pad

250* 10 25,000 x g 0.5, 1.0, 1.5, 2.0 ml clear only

02-681-452 1.5ml capless graduated non sterilemicrocentrifue tubes

1000 10 26,000 x g 0.1, 0.5, 1.0, 1.5 ml clear only

02-681-453 2.0ml capless graduated non sterilemicrocentrifue tubes

1000 10 25,000 x g 0.5, 1.0, 1.5, 2.0 ml clear only

For more information on our full range of centrifuge solutions, visit www.thermo.com/centrifuge

EQUIPMENT: CENTRIFUGES, SPECTROPHOTOMETER, FREEZERS3

7

Choose your equipment

Highlights

• Choice of 17,000 x g and 21,100 x gin both ventilated and refrigeratedmodels

• 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 ClickSealbio-containment lidcomes standard

• Practical lid design allows rotor toaccommodate SurePrep spincolumns

• Fast acceleration/deceleration: 12 sec• Five rotors to choose from, including

a unique dual-row rotor that spins 18each of 1.5/2.0 ml and 0.5 ml tubes atthe same time – eliminating the needfor adapters

• The large LED display and intuitivecontrols make operation easy; apulse capability allows you to togglebetween RPM and RCF

Centrifuges for Separating RNAIdeally suited to RNA applications, the ThermoScientific 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-refrigeratedmodels. The 24-place rotor is uniquely designed to accommodate the Fisher Scientific SurePrep spincolumns. Fast acceleration and deceleration rates onall models help you process samples quickly, and awide selection of rotors gives you added flexibility for other microcentrifuge protocols.

Table 5: Thermo Scientific Sorvall LegendMicrocentrifuge Ordering Information

* All catalog numbers include 24-place microtube rotor with ClickSeal bio-containment lid.

Cat. No. Description* Max RPM / RCF

Ventilated

75002431 Sorvall Legend Micro 17 120V 60 Hz

13,300 / 17,000

75002436 Sorvall Legend Micro 21 120V 60 Hz

14,800 / 21,100

Refrigerated

75002441 Sorvall Legend Micro 17R120V 60 Hz

13,300 / 17,000

75002446 Sorvall Legend Micro 21R120V 60 Hz

14,800 / 21,100

Table 6: Thermo Scientific Spectronic BioMate 3 UV/VisSpectrophotometer Ordering Information

EQUIPMENTSpectrophotometers for Quantifying RNAThe concentration and purity of an RNA solution can be determined by measuring its absorbanceat 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 measureconcentrations of dsDNA, ssDNA, RNA, and oligonucleotides.

To determine the RNA concentration in µg/ml, multiply the A260 by the dilution factor and theextinction 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.

8

Freezers for Storing RNA Purified RNA in elution buffer from SurePrep kits may be stored at -20°C for a few days, but forlong term storage -70°C is recommended. Thermo Scientific Revco PLUS -86°C Freezers deliverthe 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

Spectronic BioMate 3

For more information on our full range of cold storage solutions, visit www.thermo.com/cold

Cat. No. Description

14-298-2081 100-240V, 50/60Hz, US line cord

14-298-2082 100-240V, 50/60Hz, US line cord/built-in printer

14-298-2073 100-240V, 50/60Hz, US line cord6-position automatic cell holder

14-298-2074 100-240V, 50/60Hz, US line cord 6-position automatic cell holder/built-in printer

14380505 BioMate 3 and 0.2mm nanoCell bundle

Cat. No. DescriptionVolumeLiters (cu. ft.)

13-989-721 Revco Ultima PLUS 691 (24.2)

13-989-719 Revco Ultima PLUS 572 (20.2)

13-989-717 Revco Ultima PlUS 487 (17.2)

13-989-715 Revco Elite PLUS 691 (24.2)

13-989-706 Revco Elite PLUS 572 (20.2)

13-989-713 Revco Value PLUS 691 (24.4)

4 DOWNSTREAM APPLICATIONS FOR GENE EXPRESSION ANALYSIS

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 theabundance of a particular RNA species or the structural detail of an RNA transcript.

Choose your downstream application

9

Thermo ScientificHybaid Shake & Stack

Hybridization Oven

Thermo ScientificOwl Easycast B1 Gel Box

Thermo ScientificPrecision General

Purpose Water Baths

For more information on the full range of Thermo Scientific laboratory equipment solutions, visit www.thermo.com.

Gel ElectrophoresisAfter RNA is extracted fromthe sample, it is usually firstseparated by electrophoresison a denaturing agarose gel to verify its integrity or to separate various RNA speciesfor further analysis. This separation can be carried out with a submarine electrophoresis gel box – suchas the Thermo Scientific OwlEasycast B1 gel box – and lowvoltage power supplies. In addition, Fisher BioReagentsoffers functionally-testedreagents and buffers foragarose gel electrophoresis.

Northern BlotHybridizationNorthern blot analysis is used to determine the size and amount of a particularRNA species. The hybridizationprocess is carried out in a temperature-controlled chamber such as the ThermoScientific Hybaid Shake &Stack. This oven providesunmatched reliability, accuracyand uniformity to ensurereproducible results. FisherBioReagents offers functionallytested reagents and buffers for the hybridization process.

Ribonuclease ProtectionAssay (RPA)RNase protection assays are used to measure the abundance of specific mRNAs.RPA is superior to Northernblots for detecting and quantifying low-abundanceRNAs. In the RPA process, the RNA target and an RNAprobe are hybridized in solution. The hybridizationmixture is placed in a temperature-controlled waterbath set at the annealing temperature. Our ThermoScientific Precision GeneralPurpose Water Baths provideexceptional performance andreliability for the RPAhybridization process.

10

SurePrep RNA Purification Troubleshooting Guide

Problem Possible Cause Solution and Explanation

Poor RNA recovery

Incomplete lysis of cells or tissue Ensure that the appropriate amount of Lysis Solution was used for the amount of cells or tissue.

Column has become clogged Do not exceed the recommended amounts of starting biological material. Theamount of starting material may need to be decreased if the column showsclogging below the recommended levels. See also “Clogged Column” below.

An alternative elution solution was used

It is recommended that the Elution Buffer supplied with this kit be used for maximum RNA recovery.

Ethanol was not added to the lysate Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column.

Ethanol was not added to the Wash Solution

Ensure that 50 mL of 95% ethanol is added to the supplied Wash Solution prior to use.

Low RNA content in cells or tissues used

Different tissues and cells have different RNA contents, and thus the expectedyield of RNA will vary greatly from these different sources. Please check literature to determine the expected RNA content of your starting material.

Cell culture: Cell monolayer notwashed with PBS

Ensure that the cell monolayer is washed with the appropriate amount of PBS in was order to remove residual media from cells.

Yeast: Lyticase was not added resuspension buffer

Ensure that the appropriate amount of lyticase is added when making the to the Resuspension Buffer.

Bacteria and yeast: All traces media not removed

Ensure that all media is removed prior to the addition of the lysis solutionthrough of aspiration.

Clogged column

Insufficient solubilization of cells or tissues

Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue.

Maximum number of cells or amount of tissue exceeds kit specifications

Refer to specifications to determine if amount of starting material falls within kit specifications

Clarified lysate was not used for the binding step

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.

High amounts of genomic DNA present in sample

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.

Centrifuge temperature too low 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.

RNA is degraded

RNase contamination RNases may be introduced during the use of the kit. Ensure proper proceduresare followed when working with RNA. Please refer to “Working with RNA” in Section I of this user guide.

Cell lysis procedure not performedquickly enough

In order to maintain the integrity of the RNA, it is important that the procedurebe performed quickly. This is especially important for the Cell LysatePreparation Step in the Animal Tissue protocol, since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized.

Improper storage of the purified RNA 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.

Frozen tissues or cell pellets wereallowed to thaw prior to RNA isolation

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.

Starting material may have high RNase content

For starting materials with high RNase content, it is recommended that 2-mercaptoethanol be added to the Lysis Solution.

Lysozyme or lyticase used may not be RNase-free

Ensure that the lysozyme and lyticase being used with this kit is RNase-free, in order to prevent possible problems with RNA degradation.

RNA does not performwell in downstream applications

RNA was not washed 3 times with the provided Wash Solution

Traces of salt from the binding step may remain in the sample if the column isnot washed 3 times with Wash Solution. Salt may interfere with downstreamapplications, and thus must be washed from the column.

Ethanol carryover 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.

Genomic DNA contamination

Large amounts of starting material used

Perform RNase-free DNaseI digestion on the RNA sample after elution to remove genomic DNA contamination.

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