Quality-RNA.pdf

Sample & Assay Technologies

Quality RNA

High-performance RNA for gene expression analysis

2 www.qiagen.com RNA Brochure 06/2010

Table of contents

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

Introduction 3

Sample collection and stabilization 4

Sample disruption and homogenization 7

Purification of total RNA 9

Removal of genomic DNA contamination 14

Purification of microRNA 16

DNA, RNA, and protein from the same sample 18

Automated purification of RNA 20

Purification of viral nucleic acids 23

RNA quality control 24

Ordering information 26

Sample type Page

Bacteria 4 – 6

Blood 4 – 6, 13, 17

Bone marrow 4 – 6

Cells 4 – 6, 11, 15, 17, 19

Fine needle aspirates 15, 19

Fungi 13

FFPE tissue sections 12, 17

Laser-microdissected cyrosections 15, 19

Plasma 13, 17, 23

Plants 13

RNA cleanup 13

Saliva 4 – 6

Serum 13, 17, 23

Tissues 4 – 6, 11, 12, 15, 17, 19

Yeasts 11

Find the right QIAGEN products for your sample type!

RNA Brochure 06/2010 www.qiagen.com 3

1. Introduction

Reliable results in gene expression analysis applications such as real-time RT-PCR and microarray analysis depend strongly on the quality of the RNA sample used. To ensure success in your experiments, QIAGEN provides a comprehensive range of RNA sample technologies that deliver high-quality RNA from biological samples. Each step of the RNA isolation procedure (Figure 1) is streamlined and standardized:

QIAGEN also provides a broad range of technologies for gene expression analysis using end-point or real-time RT-PCR. Successful results are achieved at the first attempt using optimized master mixes and gene- and pathway-specific assays. Dedicated instruments for reaction setup and for end-point or real-time detection of PCR products are also available.

■n Sample collection and stabilization: Convenient reagents stabilize RNA immediately upon sample collection, preventing RNA degradation and changes in transcript levels. Sample disruption and homogenization:■n Robust instruments disrupt and homogenize biological samples at a range of throughputs to release RNA and reduce sample viscosity. RNA purification:■n A wide range of kits deliver manual or automated purification of high-quality RNA from a variety of sample types and sample sizes and at different throughputs.

Introduction

Disruption and homogenization

Total RNA purification

Gene expression analysis

Collection and stabilization

Figure 1. Workflow for preparing RNA for gene expression analysis.

Find out more about QIAGEN® technologies for gene expression analysis at www.qiagen.com/GEX

Chaptertitle X.XX





2. Sample collection and stabilization

Find out more about stabilization technologies at www.qiagen.com/Stabilization

Once a biological sample is harvested, its RNA becomes extremely unstable. The RNA is degraded by RNases, and gene induction or downregulation triggered by sample manipulation will also occur (Figure 2). Immediate stabilization of cellular RNA to preserve mRNA levels is critical for accurate gene expression analysis.

Sample collection and stabilization

True in vivo transcript level

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Sample harvest and processing

Enzymatic degradationand/or down-regulation


Induction

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Figure 2. Changes in mRNA levels following sample harvesting. Once a biological sample is harvested, its RNA becomes extremely unstable. Sample handling can cause gene induction or downregulation, leading to an increase or decrease in mRNA levels. mRNA levels can also decrease due to degradation of RNA by RNases.

QIAGEN provides a broad range of reagents for convenient stabilization of RNA in cells, tissues, blood, bone marrow, bacteria, and saliva at room temperature (Table 1). The use of hazardous liquid nitrogen or dry ice to freeze samples is avoided. Samples are simply submerged in the reagents to immediately preserve the gene expression profile, and can then be conveniently handled and transported at ambient temperature prior to RNA purification. For further convenience, stabilization reagents are also available as part of QIAGEN kits for RNA purification.


Chaptertitle X.XX2. Sample collection and stabilization

Table 1. Solutions for sample collection and stabilization

Sample type Products for sample collection and stabilization Stability of stabilized sample

Tissues* Allprotect Tissue Reagent (for DNA/RNA/protein stabilization)

RNAlater® RNA Stabilization Reagent (for RNA stabilization)

RNAlater TissueProtect Tubes (for tissue collection and RNA stabilization)

RNeasy® Protect Mini and Midi Kits (for RNA stabilization and purification)

Allprotect stabilized samples: 7 days at 15 – 25°C

12 months at 2 – 8°C

Longer periods at – 20°C or – 80°C

RNAlater stabilized samples: 7 days at 15 – 25°C

4 weeks at 2 – 8°C


Cells RNAprotect® Cell Reagent (for RNA/DNA stabilization)

RNeasy Protect Cell Mini Kit (for RNA/DNA stabilization and RNA purification)

7 days at 15 – 25°C



Bacteria

RNAprotect Bacteria Reagent (for RNA stabilization)

RNeasy Protect Bacteria Mini Kit (for RNA stabilization and purification)

3 hours at 15 – 25°C

2 weeks at – 20°C

4 weeks at – 70°C

Saliva RNeasy Protect Saliva Mini Kit (for RNA/DNA stabilization and RNA purification)

14 days at 15 – 25°C



Animal blood

RNeasy Protect Animal Blood System, which comprises: RNAprotect Animal Blood Tubes (for blood collection and RNA stabilization)

RNeasy Protect Animal Blood Kit (for RNA purification)

miRNeasy Protect Animal Blood Kit (for microRNA purification)†‡

48 hours at 15 – 25°C


At least 3 months at – 20°C or – 80°C

Human blood PAXgene® Blood RNA System, which comprises: PAXgene Blood RNA Tubes (for blood collection and RNA stabilization)

PAXgene Blood RNA Kit (for RNA purification) PAXgene Blood miRNA System, which comprises: PAXgene Blood RNA Tubes (for blood collection and RNA stabilization)

PAXgene Blood miRNA Kit (for microRNA purification)†

3 days at 18 – 25° C

At least 50 months at – 20°C or – 70°C

Human bone marrow

PAXgene Bone Marrow RNA System, which comprises: PAXgene Bone Marrow RNA Tubes (for bone marrow collection and RNA stabilization)

PAXgene Bone Marrow RNA Kit (for RNA purification)

3 days at 18 – 25°C

5 days at 2 – 8°C

Up to 13 months at – 20°C

PAXgene Tissue fixed tissues

PAXgene Tissue System, which comprises: PAXgene Tissue Containers (for tissue collection and preservation of histomorphology and nucleic acids)

PAXgene Tissue RNA Kit (for RNA purification)

PAXgene Tissue miRNA Kit (for microRNA purification)†

7 days at 15 – 25°C

Up to 4 weeks at 2 – 8°C

* Visit www.qiagen.com/Stabilization to find out which tissues are compatible with QIAGEN stabilization reagents. † Purifies total RNA that includes microRNA and other small RNAs. ‡ For purification of small RNA (containing microRNA) and large RNA in 2 separate fractions, the RNeasy MinElute Cleanup Kit (page 13)

is also required.


2. Sample collection and stabilization

RNA, DNA, and protein stabilizationAllprotect Tissue Reagent provides immediate stabilization of RNA, DNA, and protein in freshly harvested tissues, ensuring accurate results in genomic, transcriptomic, and proteomic applications (Figure 3, and Figures 21 – 22, page 19). After stabilization, tissues can be transported at 15 – 25°C for up to 7 days, stored at 2 – 8°C for up to 12 months, or archived at – 20°C or – 80°C. Subsequent purification of RNA/DNA or RNA/DNA/protein can be carried out using AllPrep® Kits (page 18). Alternatively, RNA, microRNA, DNA, and protein can be purified separately using RNeasy Kits (page 9), miRNeasy Kits (page 16), DNeasy®/QIAamp® Kits, and Qproteome® Kits, respectively.

RNA stabilizationConvenient RNA stabilization reagents from QIAGEN include RNAprotect Reagents for cells, bacteria, and saliva as well as RNAlater RNA Stabilization Reagent for freshly harvested tissues (Figure 23, page 20). All reagents deliver immediate RNA stabilization at room temperature and allow subsequent storage and transport of samples at ambient temperature. The reagents are also available as an integral part of RNeasy Protect Kits, providing an all-in-one solution for RNA stabilization and subsequent RNA purification (Figure 4).

For integrated sample collection, stabilization, and purification, QIAGEN provides the RNeasy Protect Animal Blood System (for animal blood) and for preservation of histomorphology, PAXgene Systems (for human blood, human bone marrow, and fixed tissues) are available. Samples are collected in tubes or containers that immediately stabilize the RNA profile (Figures 13 – 14, page 13). The samples can then be conveniently transported and stored at ambient temperature prior to RNA purification. To find out more about PAXgene products, visit www.PreAnalytiX.com.

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Figure 3. Effective RNA stabilization. Rat tissues were stored at 25°C for 2–24 hours in Allprotect Reagent or PBS prior to real-time RT-PCR analysis. Transcript levels relative to those in liquid nitrogen stabilized tissues were calculated. Changes in transcript levels were prevented by Allprotect Reagent.

Figure 4. Purification of RNA without degradation. Rat kidneys were either immediately stabilized in RNAlater Reagent (Stabilized) or left unstabilized (Unstabilized). After 0–60 minutes, RNA was purified from stabilized samples using an RNeasy Protect Kit and from unstabilized samples using a standard procedure. Purified RNA was analyzed on an agarose gel, and GAPDH expression was examined by northern blot analysis. M: markers.

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Madh7 transcript in intestine

c-fos transcript in lungA

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3. Sample disruption and homogenization





Effective disruption and homogenization of a biological sample is an absolute requirement for all RNA purification procedures. Disruption releases the RNA contained in a sample, while homogenization reduces sample viscosity to facilitate subsequent RNA purification. QIAGEN provides a range of technologies for disruption and homogenization — from QIAshredder spin columns for fast and simple homogenization of cell lysates to TissueRuptor® and TissueLyser systems for mechanical disruption and homogenization of tougher samples at a range of throughputs (Figures 5 – 7, and Figure 23, page 20). TissueRuptor and TissueLyser systems deliver fast and effective disruption, and replace tedious and time-consuming methods such as manual disruption using a mortar and pestle.

Sample disruption and homogenization

Find out more about disruption technologies at www.qiagen.com/Disruption

Figure 5. Effective tissue disruption. Various rat tissues were disrupted using the TissueLyser LT or TissueLyser II. RNA was purified from 20 mg samples on the QIAcube® using the RNeasy Fibrous Tissue Mini Kit (skin, heart, and lung) or RNeasy Lipid Tissue Mini Kit (brain). RNA was eluted in a volume of 50 µl, and concentration was determined using a spectrophotometer.

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TissueLyser LT TissueLyser II


3. Sample disruption and homogenization

Mechanical disruption of single samplesThe TissueRuptor is a handheld device that provides simultaneous disruption and homogenization using TissueRuptor Disposable Probes, which contain a blade that rotates at very high speeds. As the probes are both disposable and transparent, the risk of cross-contamination is minimized and the sample disruption process can be visually monitored. Use of disposable probes also saves time as there is no need to clean the same probe after disrupting each sample.

Mechanical disruption of multiple samplesTissueLyser instruments are bead mills that simultaneously disrupt and homogenize samples through high-speed shaking with grinding beads in plastic tubes. Using an adapter that holds several tubes, the instruments disrupt multiple samples at the same time. The TissueLyser LT processes up to 12 samples per run, providing a convenient and time-saving alternative to tedious manual disruption methods such as the use of a mortar and pestle. Due to its throughput capacity, the TissueLyser LT perfectly complements the QIAcube workstation for preparation of up to 12 samples in parallel. For higher throughput needs, we offer the TissueLyser II which provides efficient disruption of up to 192 samples in parallel.

TissueRuptor Hand-held rotor–stator homogenizer for disruption of:

n Human/animal tissues

n Plant tissues

Accessories

n TissueRuptor Disposable Probes

TissueLyser LT *

Compact, cost-effective bead mill for disruption of:


n Plant tissues

n Bacteria

n Yeast

Accessories

n TissueLyser LT Adapter, 12-Tube

n TissueLyser Single-Bead Dispensers

n Stainless Steel Beads

n Sample Tubes

TissueLyser II

Medium- to high-throughput bead mill for disruption of:


n Plant tissues

n Bacteria

n Yeast

Accessories

n TissueLyser Adapter Set 2 x 24

n TissueLyser Adapter Set 2 x 96

n TissueLyser Single-Bead Dispensers

n TissueLyser Dispensers, 96-Well

n Stainless Steel and Tungsten Carbide Beads

n Sample Tubes

Figure 6. Instruments for mechanical disruption and homogenization. * TissueLyser instruments must be used in combination with the recommended adapter (sold separately).

TissueLyser LT

TissueLyser II

Maize

T A C M R

Arabidopsis Tabacco

Figure 7. Intact RNA from plant tissues. Various plant tissues were disrupted using the nA TissueLyser LT or nB TissueLyser II. RNA was then purified using the RNeasy Plant Mini Kit and analyzed on an agarose gel. The sharp ribosomal bands indicate purification of intact RNA. T: Tomato; A: Arabidopsis; C: Cotton; M: Maize; R: Rape.

A

B

1 sample /run Up to 12 samples /run Up to 48 or 192 samples per run


4. Purification of total RNA

When purifying RNA, it is important to use a method that maintains RNA integrity and removes contaminants. Degradation of RNA makes reliable analysis of gene expression impossible, while the presence of contaminants in the purified RNA can inhibit enzymes in downstream applications such as real-time RT-PCR and microarray analysis.

Purification of total RNA





Lyse, homogenize,and add ethanol

Bind total RNAto RNeasymembrane

Wash

Elute in small volume



Wash




Wash




Wash


Wash


Bind total RNA to RNeasy membrane

Lyse, homogenize and add ethanol

Sample

Ready-to-use RNA

RNeasy Procedure

Find out more about RNA purification at www.qiagen.com/RNA

RNeasy Kits and other QIAGEN RNA purification kits (Tables 2 – 3) overcome these challenges through the combination of a specialized lysis buffer and silica-membrane technology. Biological samples are first lysed in a lysis buffer that contains a guanidine salt, which fully denatures RNases to prevent RNA degradation. RNA is then specifically bound to a silica membrane, either in an RNeasy spin column or the well of an RNeasy 96 plate. Other cellular material is efficiently washed away using a series of wash buffers before pure, intact RNA is eluted in RNase-free water.


* All kits can be automated on the QIAcube (page 21), with the exception of the RNeasy Plus Micro Kit, the QIAamp Circulating Nucleic Acid Kit, and the midi and maxi kits. † See also Table 1 (page 5) for products for RNA stabilization. ‡ See also Table 4 (page 14) for total RNA purification. § The miRNeasy Mini Kit can also be used for small amounts of plasma and serum (up to 200 µl).


Table 2. Kits for total RNA purification using spin-columns*

Sample type Kit for RNA purification Capacity

Cells†‡ RNeasy Mini, Midi, and Maxi Kits

RNeasy Plus Micro and Mini Kits

Micro: < 5 x 105 cells

Mini: 10 – 107 cells

Midi: 5 x 106 – 1 x 108 cells

Maxi: 5 x 107 – 5 x 108 cells

Easy-to-lyse tissues (e.g., kidney, liver, spleen)†‡ RNeasy Mini, Midi, and Maxi Kits

RNeasy Plus Micro and Mini Kits

Micro: < 5 mg tissue

Mini: 0.5 – 30 mg tissue

Midi: 20 – 250 mg tissue

Maxi: 150 mg – 1 g tissue

Fiber rich tissues (e.g., heart, muscle, skin)† RNeasy Fibrous Tissue Mini and Midi Kits Mini: 0.5 – 30 mg tissue


All types of tissue† RNeasy Lipid Tissue Mini and Midi Kits Mini: 10 – 100 mg tissue


All types of tissue (for microarray applications)† RNeasy Microarray Tissue Mini Kit 10 – 100 mg tissue

FFPE tissue sections RNeasy FFPE Kit Up to 8 x 10 µm sections, with surface area of up to 250 mm2

Microsamples (e.g., fine needle aspirates, laser-microdissected cryosections)†‡

RNeasy Plus Micro Kit < 5 x 105 cells

< 5 mg tissue

Animal blood (stabilized)† RNeasy Protect Animal Blood Kit < 100 µl or < 500 µl blood collected in RNAprotect Animal Blood Tubes

Human blood (stabilized)† PAXgene Blood RNA Kit 2.5 ml blood collected in PAXgene Blood RNA Tubes

Human blood (fresh) QIAamp RNA Blood Mini Kit < 1.5 ml blood

Plasma, serum, urine, and other cell-free body fluids§ QIAamp Circulating Nucleic Acid Kit < 5 ml samples

Yeast RNeasy Mini, Midi, and Maxi Kits Mini: < 5 x 107 cells

Midi: 2 x 107 – 5 x 108 cells

Maxi: 2.5 x 108 – 2.5 x 109 cells

Plants and fungi RNeasy Plant Mini Kit 102 – 107 cells

10 – 100 mg tissue

RNA cleanup and concentration RNeasy MinElute® Cleanup Kit < 45 µg RNA

RNA cleanup RNeasy Mini, Midi, and Maxi Kits Mini: < 100 µg RNA

Midi: < 1 mg RNA

Maxi: < 6 mg RNA

Table 3. Kits for manual, high-throughput RNA purification using multiwell plates

Sample type Kit for RNA purification Capacity

Cells¶ ** RNeasy 96 and Plus 96 Kits

TurboCapture® mRNA Kits††

10 – 2 x 106 cells

5 x 105 cells (96-well format) or 1 x 105 cells (384-well format)

Tissues¶ RNeasy 96 Universal Tissue Kit 25 – 100 mg tissue

Human blood (stabilized)¶ PAXgene 96 Blood RNA Kit 2.5 ml blood collected in PAXgene Blood RNA Tubes

RNA cleanup RNeasy 96 Kit

TurboCapture mRNA Kits††

< 100 µg RNA

N/A

¶ See also Table 1 (page 5) for products for RNA stabilization. ** See also Table 4 (page 14) for total RNA purification. †† For mRNA purification.



Figure 9. Sequential PCR analysis on the same plate. mRNA was purified from HEK 293 cells using the TurboCapture 96 mRNA Kit. While the isolated mRNA was immobilized in the wells of the TurboCapture plate, cDNA was synthesized. The resulting immobilized cDNA was subjected to 3 rounds of PCR, as indicated in the figure. The wells were washed 3 times with 10 mM Tris·Cl, pH 7.5 between each PCR. Wells 2 and 3 are duplicate samples. Wells 1 are negative controls in which cDNA synthesis was performed without mRNA. (Similar bands were obtained when PCRs of p53, p21, and β-actin were performed in different orders; data not shown.)

1 2 3 1 2 3 1 2 3bp

1000 –800 –600 –500 –400 –300 –200 –

100 –

1st PCR: p53

2nd PCR: p21

3rd PCR: β-actin

Purification of RNA from cells, yeast, and easy-to-lyse tissuesRNeasy Mini, Midi, and Maxi Kits provide fast and convenient purification of high-quality RNA from cultured cells as well as yeast and easy-to-lyse tissues (Figure 8, Figure 23 on page 20, and Figure 25 on page 21). A broad range of sample sizes can be processed, as each kit is supplied with RNeasy spin columns of a particular RNA binding capacity (mini: 100 µg RNA; midi: 1 mg RNA; maxi: 6 mg RNA). For very small samples (< 5 x 105 cells or < 5 mg tissue), RNA can be purified using the RNeasy Plus Micro Kit (page 15). For high-throughput applications, the RNeasy 96 Kit purifies RNA from up to 96 cell samples in parallel using RNeasy 96 plates.

Purification of mRNA from cellsTurboCapture mRNA Kits are also available for fast and simple poly-A RNA purification at high throughputs. Cell lysates are added to the wells of a TurboCapture plate (96 or 384 wells), and mRNA is allowed to hybridize to the immobilized oligo-dT in each well. Contaminants are washed away, and the isolated mRNA is then either eluted or used directly in cDNA synthesis (Figure 9).

Figure 8. Reproducible RNA yields. RNA was purified from eight HeLa cell samples (106 cells each) using either the RNeasy Mini Kit (RNeasy) or acid-phenol–extraction (Acid Phenol). nA Absorbance measurements revealed more reproducible RNA yields with the RNeasy method. nB Agarose gel analysis confirmed the greater repro-ducibility in RNA yields with the RNeasy method (10 µg of each RNA sample was loaded; amounts were determined by absorbance measurements at 260 nm).


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* Data excerpted from Ducray, F. et al. (2008) Anaplastic oligodendrogliomas with 1p19q codeletion have a proneural gene expression profile. Molecular Cancer 7, 41.

Purification of RNA from tissuesFor difficult-to-lyse tissues, QIAGEN offers specialized kits based on proven RNeasy technology. RNeasy Fibrous Tissue Mini and Midi Kits are intended for fibrous tissues, using proteinase K to remove abundant protein prior to RNA purification on RNeasy spin columns (Figure 5, page 7). RNeasy Lipid Tissue Mini and Midi Kits (spin-column format) and the RNeasy 96 Universal Tissue Kit (96-well format) are for use with all tissues, providing phenol-based QIAzol® Lysis Reagent for rigorous tissue lysis (Figure 10).*

Purification of RNA from tissues for microarray analysisThe RNeasy Microarray Tissue Mini Kit provides the optimal solution for purifying RNA for microarray analysis (Figure 12). High yields of pure RNA free from phenol contamination are achieved using the combination of QIAzol Lysis Reagent and RNeasy spin columns. Precipitation of RNA prior to cleanup is not required, saving time and preventing potential loss of RNA.

The RNeasy FFPE Kit delivers maximal yields of usable RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections (Figure 11). The combination of lysis buffer and proteinase efficiently releases RNA from tissue sections while avoiding further RNA degradation. In addition, special lysis and incubation conditions reverse formaldehye modification of RNA.

Purification of RNA from FFPE tissues

Figure 10. Reproducible real-time RT-PCR using RNA from rat brain. RNA was purified from 48 brain samples (50 mg each) using the RNeasy 96 Universal Tissue Kit. Purified RNA samples were analyzed using a C-JUN gene expression assay (the same volume of eluate, equivalent to about 90 ng RNA, was used for each assay).

Figure 11. Recovery of all usable RNA. RNA purified from 6-month-old FFPE rat liver using the RNeasy FFPE Kit or a kit from Supplier R was analyzed on the Agilent

® 2100 Bioanalyzer. The arrow indicates small RNA fragments that were recovered only with the RNeasy FFPE Kit.

A

B

C

1p19q EGFR

Figure 12. Reliable microarray analysis. RNA was purified from 13 gliomas (4 with 1p19q codeletion, and 9 with EGFR amplification) using QIAzol/RNeasy technology, and analyzed using the GeneChip® Human Genome U133 Plus 2.0 Expression Array. Unsupervised hierarchical clustering analysis was carried out using the 1366 probe sets whose expression varied the most across all samples. Lowest and highest intensity values are represented by blue and red, respectively, and show a difference in gene expression between the 2 glioma types.*

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* For larger samples, RNA cleanup can be achieved using RNeasy Mini, Midi, and Maxi Kits and the RNeasy 96 Kit (page 11).

Cleanup and concentration of RNA

Figure 13. Effective RNA stabilization in animal blood. Rat blood was collected in RNAprotect Animal Blood Tubes or EDTA-containing tubes, and stored at 15 – 25°C for 0 minutes (t=0) to 6 hours. RNA was purified using the RNeasy Protect Animal Blood Kit, and FOS expression was analyzed in duplicate by real-time RT-PCR. The Y-axis shows CT values subtracted from those for the t=0 samples. FOS transcript levels remained stable only in the RNAprotect-stabilized samples.

Figure 14. RNA stability in human blood. Blood was stored in PAXgene Blood RNA Tubes for 0 – 3 days at 18 – 25°C, and RNA was purified using the PAXgene Blood RNA Kit. Levels of FOS transcript relative to 18S rRNA were determined by duplex, real-time RT-PCR. The graph shows the values for 10 samples (collected in duplicate) plus the means and standard deviations. Dashed lines indicate the ±3x total precision of the assay (2.34 CT).

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Figure 15. Concentration of picogram amounts of RNA. The indicated amounts of RNA from HeLa cells were concentrated using the RNeasy MinElute Cleanup Kit. Real-time RT-PCR to analyze 5 µl of each 12 µl eluate was performed using the QuantiTect

® Probe RT-PCR Kit and an ACTB (β-actin) gene expression assay.2 6 10 14 18 22 26 30 34 38

10.0009.0008.0007.0006.0005.0004.0003.0002.0001.0000.000

– 1.000

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Purification of RNA from blood, serum, and plasmaFor optimal results in purification of RNA from blood, QIAGEN offers the RNeasy Protect Animal Blood System and the PAXgene Blood RNA System (see pages 5 – 6). Both integrate blood collection with RNA stabilization and purification (Figures 13 –14). Also available is the QIAamp RNA Blood Mini Kit, which provides RNA purification from white blood cells (after erythrocyte lysis). For purification of free-circulating RNA or DNA from plasma and serum, the QIAamp Circulating Nucleic Acid Kit can be used.

Purification of RNA from plants and fungiThe RNeasy Plant Mini Kit includes an alternative lysis buffer for plants, QIAshredder spin columns for homogenizing and filtering viscous plant or fungi lysates and high-molecular–weight secondary metabolites from plant extracts, and RNeasy Mini spin columns for purifying up to 100 µg of high-quality RNA (Figure 7, page 8).

The RNeasy MinElute Cleanup Kit enables cleanup and concentration of RNA from enzymatic reactions or crude RNA preparations (Figure 15). Using specialized RNeasy MinElute spin columns, up to 45 µg RNA can be purified and eluted in a volume as low as 10 µl.*

64 ngRNA:256 ng

4 ng

16 ng16 ng

64 ng

1 ng4 ng1 ng



RNA purification with DNA removal

DNA removal and gene expression

analysis

Trace amounts of genomic DNA in an RNA sample can compromise the accuracy of sensitive applications such as real-time RT-PCR. Both RNA and DNA targets may be amplified, leading to unreliable quantification of the intended RNA target. While avoiding coamplification of DNA targets can be achieved through careful design of PCR primers, this may not always be possible. QIAGEN technologies effectively eliminate genomic DNA contamination either during RNA purification or just prior to cDNA synthesis (Table 4). In both cases, virtually all traces of genomic DNA are removed, ensuring accurate quantification of RNA targets in real-time RT-PCR.

Removal of genomic DNA contamination

5. Removal of genomic DNA contamination

Table 4. Technologies for removal of genomic DNA contamination

Technology Description

RNeasy Plus Kits RNA purification kits which use gDNA Eliminator columns or plates for rapid removal of genomic DNA

RNase-Free DNase Set Reagents for DNase digestion of RNA bound to RNeasy membranes prior to RNA elution or DNase digestion in solution

FastLane® Cell cDNA Kit Kit for preparation of cDNA directly from cell lysates; includes integrated step to eliminate genomic DNA

QuantiTect Reverse Transcription Kit Kit for preparation of cDNA from total RNA; includes integrated step to eliminate genomic DNA

Find out more about DNA elimination at www.qiagen.com/DNAremoval


5. Removal of genomic DNA contamination

RNA purification with gDNA Eliminator columnsRNeasy Plus Kits integrate fast, convenient RNA purification with effective elimination of genomic DNA contamination (Figure 16). Cells or tissues are lysed in a special buffer, which provides optimized conditions for removal of DNA by a brief centrifugation through a gDNA Eliminator spin column. Total RNA is then purified using an RNeasy spin column.

The kits are available in micro or mini format for purification of up to 45 µg or 100 µg RNA, respectively. The micro format is well suited for tiny samples, such as fine needle aspirates and laser-microdissected cryosections. The mini format can be optionally automated on the QIAcube (page 21). For RNA purification in 96-well format, the RNeasy Plus 96 Kit allows rapid genomic DNA removal using gDNA Eliminator plates.

DNase digestionThe RNase-Free DNase Set provides efficient digestion of DNA on silica membranes during RNA purification using RNeasy Kits or the QIAamp RNA Blood Mini Kit (pages 9 –13).* DNase is then efficiently removed in subsequent wash steps. The RNase-Free DNase Set can also be used to digest DNA in purified or partially purified RNA samples.

cDNA from cells with integrated genomic DNA removal The FastLane cDNA Kit provides a fast and simple procedure for preparing first-strand cDNA directly from cultured cells without RNA purification (Figure 17). To eliminate genomic DNA contamination, cell lysates are briefly treated with gDNA Wipeout Buffer prior to the start of cDNA synthesis.

The unique QuantiTect Reverse Transcription Kit provides a fast and convenient procedure for cDNA synthesis with integrated genomic DNA removal. Before starting reverse transcription, a brief incubation with gDNA Wipeout Buffer effectively eliminates genomic DNA contamination.

Figure 16. High, reproducible RNA yields and effective genomic DNA removal. Total RNA was purified from tenfold serial dilutions of Jurkat cell homogenates (5 x 105 to 5 cells) using the RNeasy Plus Micro Kit; at each dilution, RNA was purified from 4 replicates. ACTB (β-actin) transcript was detected in down to 5 cells by real-time RT-PCR using the QuantiTect Probe RT-PCR Kit, and reproducible CT values were observed at each dilution. In control reactions without reverse transcriptase (– RT), ACTB DNA was not detected after 38 cycles, indicating the absence of genomic DNA contamination.

* Generally, DNase digestion is not required for RNA purified with these kits, since the silica-membrane technology efficiently removes the majority of the DNA without DNase treatment. However, complete DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA.

cDNA from total RNA with integrated genomic DNA removal

Figure 17. Effective elimination of genomic DNA contamination. cDNA was prepared using the FastLane Cell cDNA Kit, with (+ RT) or without (– RT) reverse transcriptase. The cDNA was quantified using a gene expression assay for ACTB (β-actin). The – RT plot indicates no genomic DNA is detected.

0 5 10 15 20 25 30 35

Amplification Plot

5 cells

– RT

3.000

2.500

2.000

1.500

1.000

0.500

0.000

∆ R

n

Cycle


2 6 10 14 18 22 26 30 34 38 42

3.000

2.500

2.000

1.500

1.000

0.500

0.000

– 0.500

∆ R

n

0 4 8 12 16 20 24 28 32 36 40 44

Amplification – CF_10.03.05

– RT

+ RT

Cycle



micro RNA purification

micro RNA detection

miRNeasy Kits and other QIAGEN miRNA purification kits are a special adaptation of proven RNeasy technology, allowing purification of total RNA longer than approximately 18 nucleotides (Table 5). The purified RNA includes large RNAs, such as mRNA and rRNA, as well as small RNAs, such as microRNA (miRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA). Copurification of large and small RNAs allows real-time RT-PCR detection of both mRNA and miRNA using the miScript PCR System. For certain applications where large RNAs need to be removed, supplementary protocols are available for purification of an RNA fraction rich in small RNAs.

Purification of microRNA

6. Purification of microRNA

Table 5. Kits for miRNA purification*

Kit

Format

Starting material

Automatable on QIAcube

miRNeasy Mini Kit Spin column Cells and tissues Yes

miRNeasy 96 Kit 96-well plate Cells and tissues No

miRNeasy FFPE Kit Spin column FFPE tissue sections No

miRNeasy Protect Animal Blood Kit

† Spin column Stabilzed animal blood Yes

PAXgene Blood miRNA Kit

† Spin column Stabilzed human blood Yes

PAXgene Tissue miRNA Kit

† Spin column PAXgene Tissue fixed tissues No

QIAamp Circulating Nucleic Acid Kit Spin column Plasma, serum, urine and other cell-free body fluids No

* Protocol based on RNeasy Plus technology and does not require use of phenol (QIAzol) for purification of total RNA including miRNA (and other small RNAs) from cultured cells is available online. See www.qiagen.com/miRNA-protocol-OI.

† Kit forms part of an integrated system for sample collection and RNA stabilization and purification. For details, see pages 4–6.

Find out more about technologies for miRNA research at www.qiagen.com/miRNA


6. Purification of microRNA

Purification of miRNA from cells and tissues

The miRNeasy Mini Kit and miRNeasy 96 Kit allow efficient purification of miRNA from all types of cells and tissues (Figure 18, and Figure 26, page 21). Effective lysis, even with difficult-to-lyse tissues, and removal of contaminants by organic-phase extraction is achieved using QIAzol Lysis Reagent. RNA is then purified in spin-column format (miRNeasy Mini Kit) or in 96-well format (miRNeasy 96 Kit).

Purification of miRNA from FFPE tissuesThe miRNeasy FFPE Kit delivers maximal yields of usable miRNA from FFPE tissue sections (Figure 19). By providing special lysis and incubation conditions, the kit reverses formaldehyde modification of RNA. In addition, the lysis buffer in combination with a proteinase efficiently releases RNA from tissue sections while avoiding further RNA degradation. Highly pure RNA is then obtained using trusted RNeasy MinElute spin columns.

The QIAamp Circulating Nucleic Acid Kit enables efficient purification of circulating DNA or RNA from human plasma or serum and other cell-free body fluids. A specialized protocol provides efficient purification of small RNA, such as miRNAs.

For purification of miRNA from blood, QIAGEN offers the RNeasy Protect Animal Blood System and the PAXgene Blood miRNA System (see pages 5–6). Both integrate blood collection with RNA stabilization and purification.

Figure 18. Effective purification from a range of starting amounts. Total RNA was purified from nA 102–107 Jurkat cells using the miRNeasy 96 Kit or nB a dilution series of rat lung tissue homogenate from 20 mg to 200 ng using the miRNeasy Mini Kit. miRNA-enriched fractions (< 200 nucleotides) were also isolated from the same samples. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16.

Figure 19. Superior yields and performance. RNA was purified from FFPE sections of the indicated rat tissues using either the miRNeasy FFPE Kit or a similar kit from Supplier A. nA RNA yields were determined by measuring absorbance at 260 nm. nB Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16 using the miScript PCR System. CT values were lower after purification using the miRNeasy FFPE Kit, indicating that it purified higher amounts of miRNA than the kit from Supplier A.

Purification of miRNA from plasma, serum, and blood


A

Lung Liver

Mea

n yi

eld

µg

miRNeasy FFPESupplier A7

6

5

4

3

2

1

0

High-throughput

Cell number

A

37

32

27

22

17

121x107 1x106 1x105 1x104 1x103 1x102

CopurificationmiRNA-enriched fraction

C T v

alue

Low-throughput

Amount of tissue

B

37

32

27

22

17

12

C T v

alue

CopurificationmiRNA-enriched fraction

20 mg 2 mg 200 µg 20 µg 2 µg 200 ng

B

27

26

25

24

23

22

21

20Lung Liver

miR

-16

mea

n C T

miRNeasy FFPESupplier A

In areas of research such as systems biology where different analytes are analyzed, reliable correlation of data is only possible if the analytes are all purified from the same sample. Using AllPrep Kits, a single, streamlined protocol is followed to purify DNA/RNA, RNA/protein, or DNA/RNA/protein from an entire sample, with each biomolecule eluted in a separate fraction (Table 6). Maximal yields of each biomolecule are obtained, as there is no need to divide the sample for separation purification procedures. AllPrep Kits can be used in combination with Allprotect Tissue Reagent, which stabilizes DNA, RNA, and protein in tissue samples to ensure reliable genomic, transcriptomic, and proteomic analyses.

DNA, RNA, and protein from the same sample

7. DNA, RNA, and protein from the same sample

Table 6. Kits for purification of DNA, RNA, and protein

Kit Biomolecules purified Format Capacity

AllPrep DNA/RNA Micro Kit* DNA and RNA Spin column Up to 5 x 105 cells or 5 mg tissue

AllPrep DNA/RNA Mini Kit* DNA and RNA Spin column Up to 1 x 107 cells or 30 mg tissue

AllPrep DNA/RNA 96 Kit* DNA and RNA 96-well plate Up to 1 x 107 cells or 30 mg tissue

AllPrep RNA/Protein Kit RNA and protein Spin column N/A

AllPrep DNA/RNA/Protein Mini Kit* DNA, RNA, and protein Spin column Up to 1 x 107 cells or 30 mg tissue

* Kit can be used in combination with Allprotect Tissue Reagent (page 6), which stabilizes DNA, RNA, and protein in tissues.

Find out more about multianalyte purification at www.qiagen.com/DRP



DNA, RNA, and protein purification

Genomic, transcriptomic, and proteomic analyses


7. DNA, RNA, and protein from the same sample

Purification of DNA and RNAAllPrep DNA/RNA Kits allow the simultaneous purification of genomic DNA and total RNA from the same biological sample (Figure 20). DNA is purified using optimized buffer conditions and the novel AllPrep DNA spin column, and RNA is purified from the AllPrep column flow-through using an RNeasy spin column. The kits are available in micro and mini formats, and the mini format of the kit can be optionally automated on the QIAcube (page 21). For high-throughput purification, the AllPrep DNA/RNA 96 Kit provides purification of DNA and RNA in 96-well format.

Purification of RNA and proteinThe AllPrep RNA/Protein Kit allows the simultaneous purification of total RNA and native protein from the same cultured cell sample.* The purified protein contains all cellular protein and may be used in enzyme assays.†

The AllPrep DNA/RNA/Protein Mini Kit allows simultaneous purification of DNA, RNA, and denatured protein from the same cell or tissue sample (Figures 21–22). The kit can be used in combination with Allprotect Tissue Reagent, which delivers immediate stabilization of DNA, RNA, and protein in tissue samples for long-term storage without freezing (page 6).

Figure 21. Reliable western blotting. Rat tissues were stabilized in Allprotect Reagent, and protein was purified using the AllPrep DNA/RNA/Protein Mini Kit. Duplicates were run on an SDS-PAGE gel, followed by western blotting for ERK2.

LiverSpleen

Figure 22. Reliable real-time PCR and RT-PCR analyses. Rat tissues were stabilized in Allprotect Reagent, and DNA and RNA were purified using the AllPrep DNA/RNA/Protein Mini Kit. The CT values in real-time PCR and RT-PCR analyses were similar to those achieved with tissues stabilized in liquid nitrogen.

Figure 20. Wide dynamic range. Genomic DNA and total RNA were purified from tenfold serial dilutions of rat kidney homogenate using the AllPrep DNA/RNA Micro Kit; at each dilution, DNA was purified from duplicates, and RNA was purified from 4 replicates. nA Real-time PCR analysis of the Pgk1 gene and nB real-time RT-PCR analysis of the Jun transcript showed reliable quantification over a wide dynamic range, from 5 mg down to 50 ng tissue.

Purification of DNA, RNA, and protein

* For applications where only denatured protein is required (e.g., SDS-PAGE and Western blotting), there is a supplementary protocol for RNeasy Kits and AllPrep DNA/RNA Kits that allows the simultaneous purification of total RNA and denatured protein from cells and tissues (www.qiagen.com/literature/protocols/pdf/RY22.pdf). Before carrying out enzyme assays, the functionality of each protein of interest needs to be tested.

†

1.200

1.000

0.800

0.600

0.400

0.200

0.000

∆ R

n

0 5 10 15 20 25 30 35 40 45Cycle

— 5 mg— 500 µg— 50 µg— 5 µg— 500 ng— 50 ng

Pgk1 geneAmplification Plot

A

7.000

6.000

5.000

4.000

3.000

2.000

1.000

0.000

∆ R

n

0 5 10 15 20 25 30 35 40Cycle

— 5 mg— 500 µg— 50 µg— 5 µg— 500 ng— 50 ng

Jun transcript Amplification Plot

B


26

24

22

20

18

16

CT v

alue

Kidney Intestine Lung Kidney Intestine Lung

DNA (c-fos) RNA (Madh7)

AllprotectLiquid N2

C T v

alue

Allprotect Liquid N2



RNA purification


QIAGEN automated systems have been thoroughly tested with proven QIAGEN chemistries to ensure highly reproducible purification of high-quality RNA. All automation is backed by QIAGEN Instrument Service, which provides flexible service support agreements, dedicated application support, and tailor-made training programs.

Purification of RNA on a routine basis or from larger numbers of samples can be tedious and time-consuming, and may introduce variations in RNA yield and quality due to human error. To facilitate researchers, QIAGEN provides a range of automated systems for RNA purification at low to high throughputs — from just a few samples to 192 samples per run.

Automated purification of RNA

8. Automated purification of RNA

Find out more about QIAGEN automated solutions at www.qiagen.com/automation

Figure 23. Purification of RNA from a variety of tissues. Rat tissues (15 mg) were stabilized in RNAlater RNA Stabilization Reagent and disrupted using the TissueLyser II. RNA was purified using the RNeasy Mini Kit, either manually (Manual) or on the QIAcube (QIAcube).


80

70

60

50

40

30

20

10

0Spleen Heart Kidney Lung Thymus

RNA

yie

ld [µ

g]

QIAcubeManual


Automated purification using QIAGEN spin-column kitsThe innovative QIAcube uses advanced technology to automate sample preparation using QIAGEN spin-column kits (Table 7 and Figures 23 – 26). An integrated touch screen simplifies protocol selection, and clear on-screen messages guide you through worktable setup. All steps in the purification procedure are fully automated, and up to 12 samples can be processed per run. Prior to purification, samples can be effectively disrupted using a TissueLyser instrument (pages 7– 8). Visit www.qiagen.com/MyQIAcube to find out more about the QIAcube.

Figure 25. Comparable quality of RNA following manual or automated purification. RNA was purified from six Jurkat cell samples (106 cells each) using the RNeasy Mini Kit, either manually or on the QIAcube. nA RNA analysis on agarose gel. nB Comparison of RNA quality control parameters.

Figure 26. Reliable miRNA detection. Rat kidney (25 mg) was stabilized in RNAlater RNA Stabilization Reagent and disrupted using the TissueLyser II. Total RNA (with miRNA) was purified using the miRNeasy Mini Kit, either manually (Manual) or on the QIAcube (QIAcube). Real-time RT-PCR using the miScript PCR System was carried out to detect miR-16 and miR-25.

Table 7. RNA purification spin-column kits compatible with the QIAcube

Kit Page


RNeasy Protect Mini Kit 5 – 6

RNeasy Protect Cell Mini Kit 5 – 6

RNeasy Protect Bacteria Mini Kit 5 – 6

RNeasy Protect Animal Blood Kit 5 – 6

PAXgene Blood RNA Kit 5 – 6

RNeasy Mini Kit 11

RNeasy Fibrous Tissue Mini Kit 12

RNeasy Lipid Tissue Mini Kit 12

RNeasy Microarray Tissue Mini Kit 12

RNeasy FFPE Kit 12

QIAamp RNA Blood Mini Kit 13

RNeasy Plant Mini Kit 13

RNeasy MinElute Cleanup Kit 13

RNeasy Plus Mini Kit 15

microRNA purification

miRNeasy Protect Animal Blood Kit 5 – 6

PAXgene Blood miRNA Kit 5 – 6

miRNeasy Mini Kit 17

RNA and DNA purification

AllPrep DNA / RNA Mini Kit 19

Viral nucleic acid purification

QIAamp Viral RNA Mini Kit 23

QIAamp MinElute Virus Spin Kit 23

Figure 24. QIAcube.

Parameter QIAcube Manual

RNA yield 17.17 µg 17.23 µg

CV 2.88 2.91

A260/A280 ratio 2.06 2.01

RIN value 9.7 9.7

28S/18S rRNA ratio 1.8 1.8

RNA quality control

Agarose gel analysis

QIAcube (run 1) QIAcube (run 2) QIAcube (run 3) Manual

A

B

30

25

20

15

10

5

0miR-16 miR-25

Thre

shol

d cy

cle

QIAcubeManual



BioRobot

® Universal System

n 96-well purification using RNeasy 96 Kits

n Assay setup also possible

n Purifies RNA from cells, tissues, and blood

BioRobot MDx

n 96-well purification using RNeasy/QIAamp 96 Kits


n Purifies RNA from blood

n Purifies viral nucleic acids

EZ1® Advanced instruments

n Up to 6 or 14 samples per run

n Purification using dedicated EZ1 Kits

n Purifies RNA from cells and tissues


QIAsymphony

® SP and AS

n Up to 96 samples, in batches of up to 24, per run

n Purification using dedicated QIAsymphony Kits


n Purifies RNA from cells, tissues, and blood


Figure 27. Other automated systems for RNA purification.

Automated purification using other technologiesThe BioRobot Universal System provides high-throughput automated sample purification as well as reaction setup for gene expression analysis (Figures 27–28). Using one or two RNeasy 96 plates per run, the system purifies RNA from up to 96 or 192 samples with high levels of reliability and standardization. Find out more about the BioRobot Universal System at www.qiagen.com/goto/BioRobotUniversal.

Figure 28. Reproducible RNA purification. RNA was purified from 80 Jurkat cell samples (5 x 105 cells each) using the RNeasy 96 BioRobot 8000 Kit on the BioRobot Universal System. Purified RNA was analyzed by real-time one-step RT-PCR on the ABI PRISM® 7700 using the QuantiTect Probe RT-PCR Kit with primers and probe for the ACTB (β-actin) transcript.

For laboratories requiring automation with full process documentation, QIAGEN provides EZ1 Advanced instruments (for low-throughput purification) as well as the QIAsymphony SP and AS and the BioRobot MDx (for medium- to high-throughput purification) (Figure 27). Dedicated RNA kits for use on the EZ1 and QIAsymphony systems are available. Bar code reading enables complete tracking of samples and reagents throughout the entire purification process.

Further details about these and other QIAGEN automation can be found at www.qiagen.com/automation.

2222201816

6420

0 10 20 30 40 50 60 70 80

C T


When purifying viral RNA and DNA from plasma and serum, a major challenge is to concentrate the nucleic acids, as they may be extremely diluted in a large sample volume. QIAamp Kits allow purification of viral nucleic acids from starting volumes as high as 5 ml (Table 8). Viral RNA and DNA in samples from 0.14 ml up to 5 ml are isolated on a silica membrane in a QIAamp spin column, efficiently washed, and then eluted. Depending on experimental requirements, the elution volume can be adjusted.

Purification of viral nucleic acids

Table 8. Kits for purification of viral nucleic acids

Kit

Starting material

Nucleic acids purified

Max. sample volume (ml)

Elution volume (µl)

QIAamp Viral RNA Mini Kit*†‡ Plasma, serum, and other cell-free body fluids RNA only 0.14 60

QIAamp MinElute Virus Spin Kit*‡ Plasma, serum, and other cell-free body fluids Total nucleic acids 0.2 20 – 150

QIAamp MinElute Virus Vacuum Kit† Plasma, serum, and other cell-free body fluids Total nucleic acids 0.5 20 – 150

QIAamp UltraSens Virus Kit* Plasma and serum Total nucleic acids 1 60 – 100

QIAamp Circulating Nucleic Acid Kit† Plasma, serum, urine, and other cell-free body fluids

Either RNA or DNA 5 20 – 150

Automation of viral nucleic acid purification can be achieved using the combination of the QIAamp Viral RNA Mini Kit or QIAamp MinElute Virus Spin Kit and the QIAcube (page 21), which can process up to 12 samples per run. Other automated solutions from QIAGEN include EZ1 workstations for low-throughput purification and the QIAsymphony SP and BioRobot MDx for medium- to high-throughput purification.

9. Purification of viral nucleic acids

* Purification can be carried out using a microcentrifuge. † Purification can be carried out using a vacuum manifold. ‡ Automated purification can be carried out using the QIAcube.

Find out more about technologies for virus research at www.qiagen.com/VirusResearch

Collection Viral RNA purification

Viral RNA detection


Reliable results in real-time RT-PCR and microarray analysis depend on the quality of the RNA sample. Traditionally, RNA integrity is determined by denaturing agarose gel electrophoresis, where intact RNA is indicated by a 2:1 ratio of the bands for 28S and 18S rRNA. However, agarose gel analysis is time-consuming and also hazardous, as ethidium bromide staining is required to visualize RNA bands.

RNA quality control

10. RNA quality control

The innovative QIAxcel® system overcomes these limitations through the use of ready-to-use gel cartridges and automation of the entire electrophoresis and detection procedure (Figures 29 – 31). The optimized system processes up to 96 samples per run and provides unrivaled resolution, speed, and throughput in RNA and DNA analyses. A wide range of markers and other accessories is available.

Find out more about the QIAxcel system at www.qiagen.com/QIAxcel

Figure 29. QIAxcel system. Figure 30. QIAxcel Gel Cartridge.


10. RNA quality control

Effortless analysis and exceptional ease of useThe high detection sensitivity provided by the QIAxcel analyzer enables robust results even with low concentrations of RNA. The QIAxcel system speeds up analysis and streamlines the entire sample purification and analysis workflow. The BioCalculator software, provided with the QIAxcel system, is a powerful and user-friendly tool designed to support data collection and analysis. Data can be viewed in both electropherogram and gel image formats.


Figure 31. High data resolution using the QIAxcel System. Total RNA was purified from 15 mg thymus tissue stabilized in RNAlater RNA Stabilization Reagent using the RNeasy Mini Kit on the QIAcube (Q) or manually (M). Purified RNA was eluted in 50 µl RNase-free water, and eluates were analyzed on nA the QIAxcel System and nB the Agilent 2100 Bioanalyzer.

QIAxcel system Agilent 2100 BioanalyzerQ M Q M

Q Q

MM

A B

11. Ordering information

Ordering Information

Product Contents Cat. no.

AllPrep DNA/RNA 96 Kit (4) AllPrep DNA/RNA Micro Kit (50) AllPrep DNA/RNA Mini Kit (50) AllPrep DNA/RNA/Protein Mini Kit (50) AllPrep RNA/Protein Kit (50) Allprotect Tissue Reagent (100 ml) BioRobot MDx BioRobot Universal System EZ1 Advanced* EZ1 Advanced XL* FastLane Cell cDNA Kit (50) miRNeasy 96 Kit (4) miRNeasy FFPE Kit (50) miRNeasy Mini Kit (50) miRNeasy Protect Animal Blood Kit (50) PAXgene Blood miRNA Kit (50) PAXgene Blood RNA Kit (50) PAXgene Bone Marrow Kit (30) PAXgene Bone Marrow RNA Tubes (50) PAXgene Tissue Containers (10) PAXgene Tissue miRNA Kit (50) PAXgene Tissue RNA Kit (50) QIAamp Circulating Nucleic Acid Kit (50) QIAamp MinElute Virus Spin Kit (50) QIAamp MinElute Virus Vacuum Kit (50) QIAamp RNA Blood Mini Kit (50) QIAamp UltraSens Virus Kit (50)§ QIAamp Viral RNA Mini Kit (50) QIAcube QIAshredder (50)§ QIAsymphony SP* QIAxcel System QIAzol Lysis Reagent (200 ml) QuantiTect Reverse Transcription Kit (50)§ RNAlater RNA Stabilization Reagent (50 ml)‡ RNAlater TissueProtect Tubes (20 x 5 ml) RNAlater TissueProtect Tubes (50 x 1.5 ml) RNAprotect Animal Blood Tubes (50 x 100 µl) RNAprotect Animal Blood Tubes (50 x 500 µl)

For 4 x 96 preps For 50 preps For 50 preps For 50 preps For 50 preps For stabilization of 50 x 200 mg tissue For up to 96 samples per run For up to 96 or 192 samples per run For up to 6 samples per run For up to 14 samples per run For 50 reactions For 4 x 96 preps For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For 30 preps For collection and stabilization of 50 samples For collection, fixation, and stabilization of 10 samples For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For up to 12 samples per run For 50 preps For up to 96 samples per run For up to 96 samples per run 200 ml lysis reagent For 50 reactions For stabilization of 25 x 200 mg tissue For collection and stabilization of 20 x 500 mg tissue For collection and stabilization of 50 x 150 mg tissue For collection and stabilization of 50 samples (100 µl) For collection and stabilization of 50 samples (500 µl)

80311 80284 80204 80004 80404 76405

900600 9001094 9001410 9001492

215011 217061 217404 217004 217304 763134

762164† 762174‡ 764133 764114 765112 766134 765134

55114 57704 57714 52304 53704 52904 Varies

79654 9001297 9001421

76306 205311 76104

76163 76154 76544

76554


11. Ordering information

Product Contents Cat. no.

RNAprotect Bacteria Reagent (2 x 100 ml) RNAprotect Cell Reagent (250 ml) RNase-Free DNase Set (50) RNeasy 96 Kit (4)* RNeasy 96 Universal Tissue Kit (4)* RNeasy FFPE Kit (50) RNeasy Fibrous Tissue Midi Kit (10) RNeasy Fibrous Tissue Mini Kit (50) RNeasy Lipid Tissue Midi Kit (10) RNeasy Lipid Tissue Mini Kit (50) RNeasy Maxi Kit (12) RNeasy Microarray Tissue Mini Kit (50) RNeasy Midi Kit (10) RNeasy MinElute Cleanup Kit (50) RNeasy Mini Kit (50) RNeasy Mini Kit (250) RNeasy Plant Mini Kit (50)§ RNeasy Plus 96 Kit (12) RNeasy Plus Micro Kit (50) RNeasy Plus Mini Kit (50) RNeasy Protect Animal Blood Kit (50) RNeasy Protect Bacteria Mini Kit (50) RNeasy Protect Cell Mini Kit (50) RNeasy Protect Midi Kit (50) RNeasy Protect Mini Kit (50) RNeasy Protect Saliva Mini Kit (50) TissueLyser II TissueLyser LT TissueRuptor TurboCapture 96 mRNA Kit (5) TurboCapture 384 mRNA Kit (5)

For stabilization of bacteria For stabilization of sorted or cultured cells For 50 minipreps, 25 midipreps, or 17 maxipreps For 4 x 96 preps For 4 x 96 preps For 50 preps For 10 preps For 50 preps For 10 preps For 50 preps For 12 preps For 50 preps For 10 preps For 50 preps For 50 preps For 250 preps For 50 preps For 12 x 96 preps For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For 50 preps For up to 48 or 192 samples For up to 12 samples For individual samples For 5 x 96 preps For 5 x 384 preps

76506 76526 79254 74181 74881 74404 75742 74704 75842 74804 75162 73304 75142 74204 74104 74106 74904 74192 74034 74134 73224 74524 74624 75154 74124 74324 85300 85600 Varies 72251 72271

For complete ordering information, visit www.qiagen.com

* Kits for use on the EZ1 and QIAsymphony system and on the BioRobot Universal System are also available; please inquire. † Canada and USA. ‡ All other countries. Kit not available in all countries; please inquire. § Product also available in other sizes; please inquire.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

“RNAlater ®” is a trademark of AMBION, Inc., Austin, Texas and is covered by various U.S. and foreign patents.

Trademarks: QIAGEN®, QIAamp®, QIAcube®, QIAsymphony

®, QIAxcel®, QIAzol®, AllPrep®, BioRobot®, DNeasy®, EZ1®, FastLane®, MinElute®, Qproteome®, QuantiTect

®, RNAprotect

®, RNeasy

®, TissueRuptor

®, TurboCapture® (QIAGEN Group), ABI PRISM® (Applera Corporation), Agilent

® (Agilent Technologies, Inc.), GeneChip® (Affymetrix, Inc.), PAXgene® (PreAnalytiX, GmbH). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.


1060996 06/2010

www.qiagen.com

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