OMEGA Total RNA 提取试剂盒R6834说明书

E.Z.N.A.® Total RNA Kit ITable 3Kit Contents/Storage 6Quantification 7Disruption 8Animal Cell Protocol

.......................................................10Animal Tissue Protocol

.................................................13Spin/Vacuum 16DNase Digestion Protocol ............................................22Manual Revision: October 2009Innovations in nucleic acid isolation1

IntroductionThe E.Z.N.A.® RNA family of products is an innovative system that radically simplifies

the extraction and purification of RNA from a variety of sources. The key to this system

is that it uses the reversible binding properties of the HiBind Matrix in combination

with the speed of mini-column spin technology thereby permitting single or multiple

simultaneous processing of samples. There is no need for phenol/chloroform extractions

and time-consuming steps such as CsCl gradient ultracentrifugation, or precipitation

with isopropanol or LiCl are eliminated. RNA purified using the E.Z.N.A.® RNA Purification

System is ready for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA)

purification, nuclease protection, and in vitro E.Z.N.A.® Total RNA Kit I can purify up to 100 μg of total RNA from cultured

eukaryotic cells or tissue. Normally, 1 x 106 - 1 x 107 eukaryotic cells, or 5-30 mg of tissue,

can be processed in a single experiment. Fresh, frozen or RNALater® stabilized tissues

can be used. Lysis of cells or tissue occurs under denaturing conditions which inactivate

RNases. After the homogenization process, samples are applied to the HiBind RNA spin

column to which total RNA binds. Cellular debris and other contaminants are effectively

washed away after a few quick wash steps. High quality RNA is finally eluted in DEPC

treated water. Total RNA greater than 200 nt is isolated using this kit.

For isolating total RNA below 200 nt use the miRNA isolation Kit (R7034). For isolating

total RNA from gram positive bacteria, the recommended kit is the Bacterial RNA

Kit(R6950).While this kit may be used for the isolation of RNA from whole blood, we recommend

that you use the E.Z.N.A.® Blood RNA Kit (Product # R6814) as it is specifically designed for

effective hemolysis and hemoglobin removal, thereby giving higher RNA g CapacityEach HiBind RNA Mini column can bind approximately 100μg of RNA. Using greater than

30 mg of tissue or 1 x 107 cells is not recommended.2

Centrifugation ProtocolsAdd TRK Buffer and LyseHomogenizeAdd EthanolApply Sample to ColumnWash 3xDryEluteVacuum ProtocolAdd TRK Buffer and LyseHomogenizeAdd EthanolApply Sample to ColumnWash 3xDryElute3

Kit ContentsTotal RNA Kit IPreparationsHiBind RNA Mini Columns2 mL Collection TubesTRK Lysis BufferRNA Wash Buffer IRNA Wash Buffer IIDEPC WaterInstruction BookletR6834-0055105 mL5 mL5 mL1 mL1R6834-01

505010040 mL50 mL12 mL10 mL1R6834-50 mL200 mL50 mL40 mL1Storage and StabilityAll E.Z.N.A.® Total RNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature. During shipment, crystals or

precipitation may form in the TRK Lysis Buffer. Dissolve by warming buffer to 37°C.4

Preparing Reagents• Dilute RNA Wash Buffer II with absolute ethanol (96-100%) as follows:Kit

R6834-00R6834-01

R6834-02Ethanol To Be AddedAdd 20 mL absolute ethanolAdd 48 mL absolute ethanolAdd 200 mL absolute ethanol• Store diluted RNA Wash Buffer ll at room temperature.• Please remember to always wear gloves whenever working with RNA. This will minimize

RNase contamination. Use only clean RNase-free disposable plastic pipette tips when us-ing the supplied reagents.• Under cool ambient conditions, crystals may form in TRK Lysis Buffer. This is normal;

warm at 37°C to dissolve.• Optional: As a preparation step add 20µl of 2-mercaptoethanol (β-mercaptoethanol)

per 1 ml of TRK Lysis Buffer in order to achieve a working solution. This mixture can be

stored for 1 week at room temperature.5

Guideline for Vacuum ManifoldThe following is required for use with the Vacuum Protocol:A) Vacuum Manifold (We recommend Omega Bio-tek’s VAC-08) Other Compatible Vacuum Manifolds: Qiagen QIAvac24, Sigma AldrichVM20,

Promega Vacman®, or manifold with standard Luer connector

B) Vacuum Flask

C) Vacuum TubingD) Vacuum Source (review tables below for pressure settings)ManifoldVAC-08Recommended Pressure (mbar)-200 to -600

Conversion from millibars:Millimeters of mercury (mm Hg)Kilopascals (kPa)Inches of mercury (inch Hg)Torrs (Torr)Atmospheres (atmos)Pounds per Square Inch (psi)Multiply by:0.750.10.02950.750.0009870.0145Illustrated Vacuum Setup:Omega Bio-tek’s VAC-08 C)

Vacuum Tubing A)

Vacuum Manifold D)

Vacuum Source6 B)

Vacuum Flask

Quantification of RNAStorage of RNAprepared with the E.Z.N.A.® Total RNA Kit l is stable for more than a ed RNA can be stored at -70°C (RNase-free water). Under such conditions, RNA

Quantification of RNAabsorbance at 260 nm and 280 nm in a spectrophotometer. 1 O.D. unit measured atTo determine the concentration and purity of RNA, one should measure the260 nm corresponds to 40μg of RNA per ml. DEPC treated water is slightly acidicand can dramatically lower absorbance values. We suggest that you dilute thesample in a buffered solution (TE) for spectrophotometric analysis. The Aof pure nucleic acids is 2.0, while for pure protein is approximately 0.6. Therefore, a260/A280 ratioratio of 1.8-2.0 corresponds to 90-100% pure nucleic acid. [Phenol has a maximumabsorbance at 275 nm and can interfere with absorbance readings of DNA or r, the E.Z.N.A.® Total RNA Kit eliminates the use of phenol and avoids thisproblem.]RNA Qualityquality of RNA can be best assessed by denaturing agarose gel electrophoresis andIt is highly recommended that RNA Quality be determined prior to all analysis. Theethidium bromide staining. Two sharp bands should appear on the gel. These are the28S and the 18S (23S and 16S for bacteria) ribosomal RNA bands. If these bandappears as a smear towards lower molecular weight sized RNAs, the it is likely thatRNA has undergone major degradation during preparation, handling, or gh RNA molecules less than 200 bases in length do not efficiently bind to theHiBind matrix, a third RNA band, the tRNA band, may be visible when a largenumber of cells are ed YieldsFor animal cell yields, see page animal tissue yields, see page 14.7

Disruption and Homogenization of SamplesEfficient sample disruption and homogenization is essential for successful TotalRNA isolation. Complete cell wall and plasma membrane disruption is very important for

the release of all of the RNA contained in the sample. The purpose of homogenization is

to reduce the viscosity of the cell lysates produced by cell disruption. Homogenization

shears genomic DNA and other high molecular weight cell components thereby creating

a homogenous lysate. Incomplete homogenization will cause RNA binding to clog thus

preventing efficient RNA binding result in low or no yield.

Mortar and Pestle: Sample Disruption

Sample disruption using a mortar and pestle followed the chosen of homogenization

method:

Wear gloves, and take great care when working with liquid nitrogen.1.

2.

Excise tissue and promptly freeze in a small volume of liquid tissue with a ceramic mortar and pestle under approximately 10 ml of liquid

nitrogen. Pour the suspension into a pre-cooled 15ml polypropylene tube. The tube

must be pre-cooled in liquid nitrogen or the suspension will boil vigorously possibly

causing tissue the liquid nitrogen to completely evaporate and add TRK lysis buffer.3.

Homogenization:A) Homogenizer Spin column (Product # HCR 003)

Load the lysate into a homogenizer spin column pre-inserted into a 2ml collection

tube. Spin for two minutes at maximum speed in a micro centrifuge in order to

collect homogenized lysate.

B) Syringe and Needle

Shear High molecular-weight DNA by passing the lysate through a narrowneedle (19-21 gauge) 5-10 -Stator Homogenizer: Sample Disruption and HomogenizationUsing a rotor-stator homogenizer for sample disruption and homogenization cansimultaneously disrupt and homogenize most samples. The process usually takes less

than a minute depending on sample type. Many Rotor-stator homogenizers operate with

differently sized probes or generators that allow sample processing in 50ml Milling: Sample Disruption and HomogenizationBy using bead milling, cells and tissue can be disrupted and homogenized byrapid agitation in the presence of glass beads and a lysis buffer. The optimal amount of

glass beads to use for RNA isolation are 0.5mm for yeast/unicellular cells and 4-8mm for

animal tissue samples.8

Total RNA Kit I - Animal Cell ProtocolE.Z.N.A.® Total RNA Kit I Animal Cell ProtocolAll centrifugation steps used are preformed at room temperature.

General Protocol Equipment:• Absolute(~96-100%) Ethanol• Sterile RNase-free pipet tips and 1.5ml centrifuge tubes•

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