SIGMA - RNA prep kits & TRI Reagents 할인행사

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2006년 8월 1일 ~ 9월 30일, 단 두달간.

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RED Taq DNA Polymerase (D4309, 250 units, 정상소비자가격: \ 239,700)을 무료로 드립니다!

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RNA Purification Quick Reference Gudie
Click on the product for more information

Product	Yield	Speed	Starting Material	Product Description	Technology	Applications
GenElute™ Mammalian Total RNA Miniprep Kit	Up to 150 µg of total RNA	30 minutes	Up to 10⁷ mammalian cells or 40 mg of tissue per prep	Isolation of total RNA from mammalian cells and tissues	Silica bind & elute in a spin column format	RT-PCR, northern blots and microarray analysis
GenElute™ Bacterial Total RNA Miniprep Kit	Up to 55 µg of total RNA	Less than 60 minutes	Up to 1.5 ml culture or 5 x 10⁹ cells/ml	Isolation of total RNA from bacterial cells	Silica bind & elute in a spin column format	RT-PCR, northern blots and microarray analysis
GenElute™ Yeast Total RNA Miniprep Kit	Up to 130 µg of total RNA	Less than 60 minutes	Up to 1.5 ml culture or 5 x 10⁷ cells/ml	Isolation of total RNA from yeast	Silica bind & elute in a spin column format	RT-PCR, northern blots and microarray analysis
Spectrum™ Plant Total RNA Kit	Maximum 100 µg of total RNA	Less than 30 minutes	100 mg tissue	Isolation of total RNA from plant tissue	Silica bind & elute in a spin column format	RT-PCR, northern blots and microarray analysis
TRI® Reagent RNA Isolation Reagent	5 - 15 µg per million cells, 1 - 10 µg/mg tissue	90 minutes	Up to 100 mg tissue, 10⁷ cells, 10² cm plate area or 0.25 ml fluid and blood derivatives	Isolation of total RNA from a variety of starting materials	Acid guanidine thiocyanate phenolchloroform extraction	RT-PCR, northern blots and microarray analysis
SpyLine™ Poly A+ Capture Kit		90 minutes	96-well mammalian cell cultures containing 1,000 to 50,000 cells per well	Isolating poly A+ mRNA from cultured mammalian cells	SpyLine Poly A+ Capture Plate	RT-PCR
GeneElute™ mRNA Miniprep Kits GeneElute™ mRNA Direct Miniprep Kits	2-5 μg of mRNA from 100 μg of total RNA 5-10 μg of mRNA from 40 mg of rat liver or 107 HEK 293 cells	40 minutes for poly A+ mRNA from total RNA 60 minutes directly from cells and tissues	5-500 µg Total RNA 　 Up to 10⁷ mammalian cells or 50 mg tissue	Isolating polyadenylated mRNA from previously prepared total RNA Isolating mRNA directly from mammalian cells and tissues	oligo (dT) polystyrene beads	Northern Blotting, Expression Array, Chip Hybridizations, cDNA Synthesis, Library Construction.

GenElute™ Mammalian Total RNA Miniprep Kit

For isolation of total RNA from mammalian cells and tissues

The GenElute Mammalian Total RNA Purification Kit combines silica-membrane technology with a convenient spin column format for a rapid bind, wash, and elute method to prepare high quality total RNA (Figure 1).

Samples are lysed and homogenized in guanidine thiocyanate and 2-mercaptoethanol to release RNA and inactivate RNases. Lysates are spun through a filtration column to remove cellular debris and shear DNA. The filtrate is then applied to a high capacity silica column to bind total RNA, followed by washing and elution. Up to 150 µg of total RNA can be recovered per prep in 100 µl of water. The purified RNA is ready for Northern blot analysis (Figure 1), RT-PCR (Figure 2) and other common applications.

Features and Benefits

Purifies total RNA from up to 10⁷ cells or 40 mg of tissue per prep
Yields up to 150 μg of pure, concentrated total RNA per prep
Recover RNA from as few as 100 cells
Simple and efficient–12 to 18 preps in 30 minutes
Faster than gravity flow anion exchange methods
No cumbersome steps associated with resins and magnetic slurries
40% more purifications per kit than the leading supplier

Protocol for GenElute Mammalian
Total RNA Purification Kit

High quality RNA from various tissues and cells.

GenElute Mammalian Total RNA Gel

Figure 1. Formaldehyde-agarose gel and Northern blot of total RNA purified with the GenElute Mammalian Total RNA Purification Kit.
Upper panel: 2 µg of each RNA analyzed on a 1.2% agarose gel containing 0.6M formaldehyde.
Lower panel: Corresponding Northern blot hybridized with a ³²P labeled RNA probe for GAPDH in PerfectHyb^TM Plus hybridization buffer.
Note: The GAPDH probe detected a single mRNA band in every lane with little or no smearing. Even RNA from pancreas, which is known to have high RNase levels, is not degraded.

RNA purification using Sigma's GenElute Mammalian Total RNA Isolation Kit is suitable for RT-PCR.

Figure 2. RT-PCR analysis of RNA isolated with the GenElute Mammalian Total RNA Purification Kit. RT-PCR detection with RNA from 100 to 1,000 cells. HeLa cells were diluted to give 0, 100, 300, or 1,000 cells per tube followed by RNA purification using the GenElute Mammalian Total RNA Isolation Kit. Duplicate 10 µl samples (20%) of each preparation were treated with Amplification Grade DNase I. One of each pair was reverse transcribed with Enhanced Avian Reverse Transcriptase (+ lanes). The other was incubated under the same conditions, but without the reverse transcriptase (- lanes). PCR was performed with 2 µl (10%) from each reaction, GAPDH primers, and Taq DNA Polymerase. One-fifth of each PCR product was fractionated on a 1.5% agarose gel, and photographed after staining for 30 minutes with SybrGreen I. RT-PCR products are clearly visible for all reactions with reverse transcriptase added (+ lanes), except those with no cells (Lane 0). No PCR products are visible for reactions without reverse transcriptase added (- lanes), demonstrating that RT-PCR products with reverse transcriptase are from RNA and not from residual, contaminating DNA.

Components :

Lysis Solution
b-mercaptoethanol
Wash Solution 1
Wash Solution 2
Elution Solution
Filtration Columns with Tubes
RNA Binding Spin Columns with Tubes
Collection Tubes

Product #	Product Name	Preps	Sell Price	30% Off + Free RED Taq
RTN10	GenElute™ Mammalian Total RNA Purification	10	₩57,000	₩40,000
RTN70		70	₩386,000	₩270,000
RTN350		350	₩1,703,000	₩1,192,000

GenElute™ Bacterial Total RNA Miniprep Kit

For isolation of total RNA from bacteria cells

Sigma GenElute Bacterial Total RNA Purification Kit combines silica-membrane technology with a convenient spin column format for a rapid bind, wash and elute method to prepare high quality total RNA from both Gram-negative and Gram-positive bacteria.

Cells are lysed and homogenized in a guanidine thiocyanate-containing buffer to ensure thorough denaturing of macromolecules and inactivation of RNases. Addition of ethanol causes RNA to bind when the lysate is spun through a silica membrane in a microcentrifuge tube. After washing to remove contaminants, total RNA is then eluted. The purified RNA is ready for reverse transcription, PCR? labeling, microarray analysis, and other common applications.
Features and Benefits

Pure - High quality RNA in less than one hour
Flexible - Suitable for both Gram-positive and Gram-negative bacteria
Robust - Yields of 30 - 55 ug
Efficient - Purified RNA is ready for RT-PCR, Northern Blots, and Microarray analysis.

Superior Yields of RNA

1 2 3 4 5 6 7

Figure 1.Total RNA purified from B. subtilis using Sigma GenElute kit and Supplier "Q" kit. Lanes 1,2,3 show increasing cell mass respectively using Sigma kit. Lane 4 (center) is R7644, RNA marker. Lanes 5,6,7 show increasing cell mass respectively using Supplier "Q" kit. Sigma's kit shows increasing yields with increasing cell mass whereas the competitor kit achieves lower yields even with higher cell mass inputs.

Source		Type of Media		Amount of Source Material		OD₆₀₀		Typical RNA Yield**
Esherichia coli ATCC# 11775		LB Broth (Cat. Code L3522 )		1.0 ml		0.63		45 - 55 µg
Bacillus subtilis ATCC# 6051		LB Broth (Cat. Code L3522 )		1.33 ml		0.75		30 - 35 µg

Figure 2. Typical RNA yield with the GenElute Bacterial Total RNA Purification Kit.
*All readings were obtained using a Molecular Devices SpectraMax Plus Spectrophotometer.
**Based on performing 50 µl elutions.

High Quality RNA

Figure 3. Pure, intact E. coli RNA of high integrity is demonstrated by the Agilent 2100 Bioanalyzer electropherogram.

Product #	Product Name	Preps	Sell Price	30% Off + Free RED Taq
BTR1	GenElute™ Bacterial Total RNA Purification	10	₩47,000	₩33,000
BTR7	GenElute™ Bacterial Total RNA Purification	70	₩344,000	₩241,000

GenElute™ Yeast Total RNA Miniprep Kit

For isolation of total RNA from yeast cells

Sigma's GenElute Yeast Total RNA Purification Kit combines silica-membrane technology with a convenient spin column format for a rapid bind, wash and elute method to prepare high quality total RNA from yeast.

The kit uses yeast lysis reagents to digest cell walls and convert cells to spheroplasts. Spheroplasts are then lysed under denaturing conditions to release RNA and inactivate RNases. Addition of ethanol causes RNA to bind when the lysate is spun through a silica membrane in a microcentrifuge tube. After washing to remove contaminants, total RNA is then eluted. The purified RNA is ready for reverse transcription, PCR, labeling, microarray analysis, and other common applications.
Features and Benefits

Pure - High quality RNA in less than one hour
Straightforward - No cumbersome steps associated with resins and magnetic slurries
Robust - Yields up to 130 µg
Efficient - Purified RNA is ready for RT-PCR, Northern Blots, and Microarray analysis.

High Yields of RNA

Figure 1.Yield of RNA from S. cerevisiae: OD₂₆₀. The GenElute Yeast Total RNA Purification Kit and an RNA purification kit from Supplier "Q" were used as outlined in their technical bulletins for yeast RNA purification. S. cerevisiae was grown overnight in YPD broth at 30°C from an isolated colony on a freshly streaked YPD plate. The culture was grown until a cell mass of 0.4~0.7 was achieved as measured by OD₂₆₀ (18-24 hours).

Product #	Product Name	Preps	Sell Price	30% Off + Free RED Taq
YTR1	GenElute™ Yeast Total RNA Purification	10	₩51,800	₩36,000
YTR7	GenElute™ Yeast Total RNA Purification	70	₩267,000	₩187,000

Spectrum™ Plant Total RNA Kit

The total RNA purification method for difficult plant tissues

Few methods are capable of delivering desired amounts of total RNA from plant tissues that contain high levels of secondary metabolites. The Spectrum Plant Total RNA Kit was designed to be different. Incorporating a new, patent-pending lysis and binding chemistry, this kit is intended to purify total RNA from species and tissue where most protocols fail. Without sacrificing affordability, you can purify more total RNA from difficult tissue samples.

The Spectrum Plant Total RNA Kit employs a new lysis and binding chemistry and a convenient column-based 'bind-wash-and-elute' format to purify up to 100 µg of total RNA from 100 mg of tissue in about 30 minutes. Typical yields range from 20 — 60 µg. After grinding tissue to a fine powder in liquid nitrogen, cells are lysed and cellular debris is physically and chemically separated from endogenous RNA. RNA is then bound to a column supported silica substrate and several wash steps remove remaining contaminants. Lastly, total RNA is eluted from the column and used in typical applications, such as Northern Blots, and RT- and qRT-PCR. The Spectrum Plant Total RNA Kit contains all the reagents, buffers and solutions necessary to perform the method as described.

                                   Detailed Product Description

                                   FAQs (121 Kb PDF)

                                   Instruction Manual (86 Kb PDF)

                                   Application Poster (174 Kb PDF)

Spectrum™ Plant Total RNA Kits - Ordering Information

The Spectrum™ Plant Total RNA Kits are available in 3 different sizes. Each kit contains all reagents and components sufficient for the number of total RNA purifications indicated.

Product #	Product Name	Preps	Sell Price	30% Off + Free RED Taq
STRN10	Spectrum™ Plant Total RNA Kit	10*	₩114,000	₩80,000
STRN50		50*	₩287,000	₩201,000
STRN250		250*	₩1,196,000	₩837,000

*each preps from up to 100 mg of tissue

While the Spectrum Plant Total RNA Kit removes nearly all genomic DNA during the purification protocol, trace amounts may remain in certain preparations. Due to the sensitivity of PCR, trace amounts are still capable of providing sufficient template for Taq amplification.

A common approach to prevention is the use of DNase I to digest any genomic DNA that may be present. Sigma offer's this enzyme in two formats; one intended for use while the RNA is bound to the purification column and one intended for use in solution, after the RNA has been eluted. In general terms, on-column DNase digestion is faster and more convenient, while DNase digestion in solution is absolute. The decision of stringency required vs. preferred format should be based on experimental goals .

Product #	Product Name	Preps	Sell Price	30% Off + Free RED Taq
DNASE10	On-Column DNase I Digestion Set	sufficient for 10 preps	₩26,000	₩18,000
DNASE70	On-Column DNase I Digestion Set	sufficient for 70 preps	₩141,000	₩99,000
AMPD1	DNase I Amplification Grade	Sufficient to treat 1000 μg of RNA	₩108,000	₩76,000

It is worth mentioning that another mechanism used to prevent possible amplification of genomic DNA involves primer design (there are many good web references on this subject). One practice widely recommended is primers designed (at least one of a pair) to span exon/exon junctions.

TRI^® Reagent RNA Isolation Reagent

For isolation of total RNA from a variety of starting materials

TRI Reagent® is an improved version of the single-step total RNA isolation reagent developed by Chomczynski (1). The RNA isolation method based on this reagent is widely recognized and proven for RNA applications and is supported by a substantial publication list (2,3,4,5). It is ideal for quick, economical, and efficient isolation of total RNA or the simultaneous isolation of RNA, DNA and proteins from samples of human, animal, plant, yeast, bacterial and viral origin.

Features and Benefits

Easily scalable RNA isolation
Works with many sources: human, plant, yeast, bacterial, or viral
Better yields than traditional guanidine thiocyanate/cesium chloride methods
Three convenient formulations of TRI Reagent®

Tri Reagent Formulations

Product Name	Sample Type	Sample Volume	TRI Reagent Volume
TRI Reagent	Tissues, cultured adherent cells, cell pellets	up to 100 mg tissue, 10⁷ cells or 10² cm plate area	1 ml
TRI Reagent BD	Whole blood, plasma, serum	0.25 ml blood derivatives	0.75 ml
TRI Reagent LS	Cell suspension, CSF, amniotic fluid	0.25 ml fluid samples	0.75 ml

Product #	Product Name	Pack	Sell Price	30% Off + Free RED Taq
T9424	TRI Reagent	25 ml	₩65,000	₩46,000
		100 ml	₩193,000	₩135,000
		200 ml	₩348,000	₩244,000

T3809	TRI Reagent BD	25 ml	₩73,000	₩51,000
		100 ml	₩232,000	₩162,000
		200 ml	₩408,000	₩286,000
T3934	TRI Reagent LS	25 ml	₩123,000	₩86,000
		100 ml	₩343,000	₩240,000
		200 ml	₩571,000	₩400,000

SpyLine™ Poly A+ Capture Kit

For isolation of poly A+ mRNA from cultured mammalian cells for direct RT-PCR

The SpyLine™ Poly A+ Capture Kit offers a simple, rapid, and cost-effective method for isolating poly A+ mRNA from cultured mammalian cells for direct RT-PCR. This kit features the SpyLine Poly A+ Plate, which is a 96-well PCR plate coated with oligo(dT) for selective capture of poly A+ mRNA from crude mammalian cell lysates. RT-PCR reagents can be added directly to this capture plate for subsequent RT-PCR cycling and analysis, or the mRNA can be eluted from the plate if desired.

Lyse cells in culture plate

Transfer lysate to capture plate

Hybridize for 30-90 minutes

Wash

Perform RT-PCR directly in capture plate, or elute mRNA for other applications

Features and Benefits

Sensitive - Superior tool for knockdown measurement and gene expression analysis.
Flexible - Isolate mRNA from a wide variety of cell dilutions - even from a single cell.
Consistent - Extremely low well-to-well and plate-to-plate variability.
Automatable - 96-well plate format suitable for use on high-throughput platforms.
Convenient - Capture and amplify mRNA in a single plate.
Simple - Rapid, easy-to-follow protocol.

Superior sensitivity for siRNA knockdown

Figure 1. Target vs. dCT for SpyLine vs. Supplier Q. The figure above is a comparison of the change in Ct from the siRNA knockdown cells to the Non-Targeting siRNA cells. Plated HeLa cells were transfected with either siRNA transfection reagent mixture or non-targeting siRNA #1 reagent. After overnight incubation, the transfection media was removed, and the cells were lysed. One plate was purified by the SpyLine Poly A+ Capture Kit, while the other was purified using Supplier Q' 96-well total RNA purification kit. Quantitative RT-PCR was run on the 2 sets of purified RNA using Applied Biosystems TaqMan? Gene Expression Assays.

Flexible enough for a variety of cell dilutions

Figure 2. Ct vs. cell number for decreasing amount of cells. A series of cell dilutions was prepared, beginning with 10,000 cells and decreasing by ten-fold increments down to 1 cell. SURF-4 mRNAs were then amplified by quantitative RT-PCR.

Figure 3. Quantitative RT-PCR of mRNA isolated from a series of decreasing cell dilutions. Cell dilutions were prepared in replicate, in decreasing 10-fold increments from 10000 cells to 1 cell. SURF-4 mRNAs were then amplified by quantitative RT-PCR. As depicted above, message can be clearly detected all of the way down to a single-cell input.

Consistent results - extremely low well to well and plate to plate variability

Figure 4. Well to well variability and plate to plate variability of the SpyLine Poly A+ Capture Kit. mRNA was isolated and captured utilizing the SpyLine standard protocol. Quantitative RT-PCR was set-up in the SpyLine Poly A+ Capture plate and was run targeting SURF-4 mRNA. Comparison of the two plates shows very little variability on each plate (standard deviation ~0.5 Cts), and very little variability from plate to plate (standard deviation ~0.5 Cts).

Product #	Product Name	Plates	Sell Price	30% Off + Free RED Taq
SPY1	SpyLine Poly A+ Capture Kit	1 x 96	₩382,000	₩267,000
SPY4	SpyLine Poly A+ Capture Kit	6 x 96	₩1,378,000	₩965,000

GenElute™ mRNA Miniprep Kits

High yield isolation of mRNA from mammalian cells, tissues, or total RNA

Procedures such as cDNA synthesis, expression profiling, and others, require separation of mRNA from the vastly more abundant rRNA and tRNA. The GenElute mRNA Kits provide convenient procedures for isolating polyadenylated mRNA from previously prepared total RNA, or directly from mammalian cells and tissues.

For direct mRNA preparation, cells or tissues are disrupted with SDS/proteinase K digestion to release RNA and eliminate RNases. Both kit types use oligo dT₃₀ covalently linked to 1 µm polystyrene beads to capture polyadenylated mRNA by hybridization. The polystyrene beads remain suspended during hybridization, eliminating the need for mixing or rocking, as is common for cellulose or magnetic particles. Polystyrene was also chosen because oligo (dT) polystyrene beads yield cleaner mRNA with fewer stringent washing steps than does the more commonly used oligo (dT) cellulose (2 or 3 wash steps versus 10 or more). With the GenElute kits, mRNA-bead complexes are washed on a microcentrifuge spin filter, and eluted into 10 mM Tris-HCl, pH 7.5. mRNA prepared with either kit is suitable for a variety of downstream applications such as Northern Blotting (Figure 4), Expression Array or Chip Hybridizations, and cDNA Synthesis and Library Construction.

Protocol for GenElute
mRNA Miniprep Kits

Features and Benefits

Fast - Prep time is 40 minutes for poly A+ mRNA from total RNA or 60 minutes directly from cells and tissues.
Convenient - mRNA captured on polystyrene beads in 10 minutes, with no mixing or rocking. Beads washed on microcentrifuge spin filters.
Yield - Higher than all competing products.

RNA Yield

GenElute mRNA Yield Figure 2. Total RNA was prepared from Hek293 cells by a silica-binding method, using TriReagent®. mRNA was subsequently prepared from total RNA preparations using commercially available kits, following the procedures supplied by each vendor. RNA yield was measured using the RiboGreen^TM RNA Quantitation Kit (Molecular Probes).

Specific mRNA yield

GenElute Specific mRNA Yield Figure 3. mRNA was prepared from total RNA as in Figure 2, and twenty percent of each preparation was analyzed by Northern blot hybridization with ³²P-labeled probes. Hybridization to human HGPRT, human p53, and mouse p53 mRNAs were quantified with a Perkin Elmer Instant Imager.

mRNA is suitable for Northern Blots

GenElute mRNA Northern Blot
Figure 4. Northern blot comparison of mRNA prepared with GenElute mRNA kits from other suppliers. Duplicate mRNA samples were prepared from 5 x 10⁶ Hek293 cells (top left panels) or 25-35 mg mouse liver (top right panels) with Sigma's GenElute Direct mRNA Miniprep Kit (S) or with another commercially available direct mRNA miniprep kit (A, D, I, P, & Q). An optional release and rebind procedure (described in the Manual) was included for preparation S+: RNA was hybrid-captured onto oligo (dT) beads, released into fresh lysis solution, and re-captured onto the same beads before washing and eluting mRNA. The release and rebinding steps were omitted for S- & Sx2 preparations. Sx2 preparations were repurified after the final elution with fresh oligo (dT) beads (as described in the Manual) . Equal portions of each mRNA preparation, equal to the amount from 1 x 10⁶ cells or 10 mg liver, were evaluated by Northern blot hybridization with ³²P-labeled RNA probes for p53, hypoxanthine-guanine phosphoribosyl transferase (HGPRT), c-myc, and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.

For the bottom panels, total RNA was first prepared from Hek293 cells and from mouse liver. Duplicate mRNA samples were then prepared from 100 µg of these total RNA preparations with Sigma's GenElute mRNA Miniprep Kit (S) or with another commercially available mRNA miniprep kit (Q, A, I, P, D). Samples Sx2 were repurified after elution. Twenty percent of each preparation was evaluated by Northern blot as above. In lanes T, 5 and 2 µg of the original total RNA from cells or 10 and 5 µg of total RNA from liver were analyzed for comparison.

Product #	Product Name	Starting Material	Preps	Sell Price	30% Off + Free RED Taq
MRN10	GenElute mRNA Miniprep Kit	5-500 µg Total RNA	10	₩157,000	₩110,000
MRN70	GenElute mRNA Miniprep Kit	5-500 µg Total RNA	70	₩945,000	₩662,000
DMN10	GenElute Direct mRNA Miniprep Kit	Up to 10⁷ mammalian cells or 50 mg tissue	10	₩161,000	₩113,000
DMN70	GenElute Direct mRNA Miniprep Kit	Up to 10⁷ mammalian cells or 50 mg tissue	70	₩988,000	₩692,000