Sequencing Sample Prepand Gene Expression Analysis

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DNA-Seq

Nextera™ DNA Sample Prep KitsSequencer-ready libraries in under 2 hours, from only nanograms of DNA

Nextera technology simultaneously fragments and tags DNA in a single-tube reaction, and offers significant advantages over current library preparation methods:

• Sequencer-ready libraries in under 2 hours.• Incorporate platform-specific tags and optional barcodes.• Library complexity, coverage, accuracy, and bias comparable to control libraries

prepared using mechanical and other enzyme-based fragmentation methods.• Validated on multiple second-generation sequencing platforms, including Roche

454™ and Illumina® GAII and HiSeq™ 2000.

Nextera libraries have been successfully sequenced with numerous sample types, including:

• Human genomic DNA • Fosmid clones• E. coli genomic DNA • cDNA• Soy genomic DNA • LR-PCR amplicons• HIV-1 cDNA amplicons • Phage DNA• Drosophila genomic DNA • and more • Plasmids

ProcessingStep StandardMethod(~µg) NexteraMethod(<50ng)

Fragmentation ✓ (15-30 min)

Add Nextera Enzyme Mix (5 min)

Collection ✓ (15 min)

Concentration ✓ (15 min)

Size Selection ✓ (60 min)

End-Repair ✓ (60 min)

Clean-Up ✓ (15 min)

A-Tailing +/- (30 min)

Adaptor Ligation ✓ (60 min)

Clean-Up ✓ (15 min) (15 min)

Library Enrichment Variable (~60 min) PCR (~60 min)

Total Time: ~6 Hours <2 Hours

Blue color indicates steps where the sample is transferred to another tube.

Table1.ComparisonofconventionalandNextera™librarypreparationworkflows.

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DNA-Seq

Cat.# Quantity

Nextera™ DNA Sample Prep Kit (Illumina®-Compatible) GA09115 5 ReactionsGA091120 20 ReactionsGA0911-50 50 ReactionsGA0911-96 96 Reactions

Nextera™ DNA Sample Prep Kit (Roche Titanium-Compatible)NT09115 5 reactionsNT091120 20 reactionsNT0911-50 50 reactionsNT0911-96 96 reactionsContents: Nextera Enzyme Mix (Illumina®-compatible), 5X Nextera Reaction Buffer (LMW), 5X Nextera Reaction Buffer (HMW), 50X Nextera Primer Cocktail (Illumina®-compatible), 50X Nextera Adaptor 2 (Illumina®-compatible), 200X Nextera Read 1 Primer, 200X Nextera Read 2 Primer, 200X Nextera Index Read Primer, Nextera Control DNA, and 2X Nextera PCR Buffer.

Nextera™ Barcodes (optional)

Illumina®-CompatibleGABC0950 12 Bar Codes

Roche Titanium-CompatibleNTBC0950 12 Bar CodesNote: The barcoding kits are optional. Each kit contains 12 barcodes, and each barcode can be used to prepare up to 50 barcoded libraries.

Additional Nextera Kits:

Nextera™ PCR EnzymeEM091120 20 ReactionsEM091150 50 ReactionsEM0911-96 96 ReactionsImportant: Nextera PCR Enzyme must be purchased separately when using any of the Nextera DNA Sample Prep kits.

Illumina now sells and supports a new version of Nextera kits. These new kits offer several advantages over the current Epicentre kits, which will be discontinued effective December 31, 2011.

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DNA-Seq

End-It™ DNA End-Repair KitRepair DNA ends for adaptor ligation

The End-It DNA End-Repair Kit converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA. The end-repaired DNA can be used for ligation into a cloning vector or ligation of next-generation sequencing adaptors, using the Fast-Link™ DNA Ligation Kit. The high specific activity of the End-Repair Enzyme Mix provides complete conversion of protruding ends to 5′-phosphorylated, blunt-ended DNA.

The End-It DNA End-Repair Kit contains reagents for 20 or 50 end-repair reactions (repair of up to 100 µg or 250 µg of genomic DNA, respectively).

Cat.# Quantity

End-It™ DNA End-Repair KitER0720 20 ReactionsER81050 50 ReactionsContents: End-Repair Enzyme Mix, End-Repair 10X Buffer, dNTP Solution, ATP.

Fast-Link™ DNA Ligation KitLigate next-generation sequencing adaptors to DNA

The Fast-Link DNA Ligation Kit uses a high-quality ligase (Fast-Link T4 DNA Ligase), to provide extremely rapid, high-efficiency DNA ligation. Cohesive-end ligations can be performed in 5 minutes at room temperature. In contrast to other ligases, it is not necessary to desalt Fast-Link ligation reactions prior to transformation of electrocompetent or chemically competent cells. The Fast-Link Kit can be used for routine and high-throughput DNA cloning.

Cat.# Quantity

Fast-Link™ DNA Ligation KitLK11025 25 LigationsLK0750H 50 LigationsLK6201H 100 LigationsContents: Fast-Link DNA Ligase, Fast-Link 10X Ligation Buffer, 10 mM ATP.

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DNA-Seq

CircLigase™ and CircLigase II ssDNA LigasesCircularize ssDNA templates

CircLigase ssDNA Ligase* is a thermostable ATP-dependent ligase that catalyzes intramolecular ligation (i.e., circularization) of ssDNA templates having a 5′-phosphate and a 3′-hydroxyl group. In contrast to other ligases, CircLigase ssDNA Ligase ligates ends of ssDNA in the absence of a complementary sequence. The enzyme is therefore useful for making circular ssDNA molecules from linear ssDNA. Circular ssDNA molecules can be used as substrates for rolling-circle replication or rolling-circle transcription.

CircLigase II ssDNA Ligase* has greater activity and a new Reaction Buffer for improved ligation efficiency.

Unit Definition: One unit of CircLigase enzyme converts 1 pmol of a linear 5′-phosphorylated CircLigase Standard 55-mer Oligo into an Exonuclease I–resistant circular form in 1 hour at 60°C under standard assay conditions.*Covered by intellectual property rights licensed to Epicentre.

Cat.# Quantity

CircLigase™ ssDNA LigaseCL4111K 1,000 UnitsCL4115K 5,000 UnitsContents: CircLigase ssDNA Ligase, CircLigase 10X Reaction Buffer, 1 mM ATP, 50 mM MnCl2, CircLigase ssDNA Control Oligo, Sterile Water.

CircLigase™ II ssDNA LigaseCL9021K 1,000 UnitsCL9025K 5,000 UnitsContents: CircLigase II ssDNA Ligase, CircLigase II 10X Reaction Buffer, 50 mM MnCl2, CircLigase ssDNA Control Oligo, Betaine, Sterile Water.

DisplaceAce™ DNA Polymerase Rapid DNA sequencing of GC-rich regions

DisplaceAce DNA Polymerase* is a recombinant DNA polymerase modified to remove 5′→3′ exonuclease activity. It has strong strand-displacing DNA polymerase activity, similar to that of Bacillus DNA polymerases. The DNA-dependent DNA polymerase activity is optimal at approximately 65°C. It also has RNA-dependent DNA polymerase activity. The enzyme can be inactivated by incubation at 80°C for 20 minutes.

Unit Definition: One unit converts 10 nmol of dNTPs into acid-insoluble material in 30 minutes at 65°C under standard assay conditions.*Covered by issued and/or pending patents.

Cat.# Concentration Quantity

DisplaceAce™ DNA PolymeraseD090710K 100U/ul 10,000 Units

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DNA-Seq

Plasmid-Safe™ ATP-Dependent DNaseRemove bacterial chromosomal DNA from plasmid preps

Plasmid-Safe ATP-Dependent DNase selectively removes contaminating bacterial chromosomal DNA from plasmid, cosmid, fosmid, and BAC clones or vector preparations. Such preparations are frequently contaminated with fragments of bacterial genomic DNA generated during alkaline lysis. Other purification options, such as spin-columns or even CsCl centrifugation, do not effectively remove these contaminants and require further purification steps.

Plasmid-Safe ATP-Dependent DNase digests linear dsDNA to deoxynucleotides at slightly alkaline pH and, with lower efficiency, closed-circular and linear ssDNA. The enzyme has no activity on nicked or closed-circular dsDNA or supercoiled DNA.

Unit Definition: One unit of Plasmid-Safe DNase converts 1 nmol of deoxynucleotides in linear T7 DNA into an acid-soluble form in 30 minutes at 37°C under standard assay conditions. Three units will digest 1 µg of linear T7 DNA in 30 minutes at 37°C.

Cat.# Concentration Quantity

Plasmid-Safe™ ATP-Dependent DNaseE3101K 10 U/µl 1,000 UnitsE3105K 10 U/µl 5,000 UnitsE3110K 10 U/µl 10,000 UnitsIncludes Plasmid-Safe 10X Reaction Buffer and 25-mM ATP Solution.

Proteinase K Remove protein after selection in ChIP protocols

Proteinase K is used to digest protein and remove contamination from preparations of nucleic acid. The addition of Proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification. Proteinase K remains active under denaturing conditions, such as the presence of SDS or urea, chelating agents (EDTA, sulfhydryl reagents), and trypsin or chymotrypsin inhibitors.

Cat.# Concentration Quantity

Proteinase KMPRK092 50 µg/µl 2 ml

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DNA-Seq

Exonuclease III, E. coli Produce nested deletions for site-directed mutagenesis

Exonuclease III digests ds DNA in a 3′→5′ direction from a nick, a blunt end, or 3′-recessed end, producing stretches of ssDNA on the opposite strand. Under defined reaction conditions, DNA degradation by Exonuclease III proceeds at a uniform rate yielding predictable and reproducible digestion results.

Exonuclease III is not active on 3′-protruding ends of four bases or more in length, ssDNA, or on thioester-linked nucleotides.The enzyme also has intrinsic RNase H, 3′-DNA phosphatase, and apurinic DNA endonuclease activities.

Unit Definition: One unit of Exonuclease III catalyzes the release of 1 nmol of acid-soluble nucleotides from ds calf thymus DNA in 30 minutes at 37°C under standard assay conditions.

Cat.# Concentration Quantity

Exonuclease III, E.coliEX4405K 200 U/µl 5,000 UnitsEX4425K 200 U/µl 25,000 UnitsIncludes 10X Reaction Buffer.

Exonuclease I, E. coliRemove excess ssDNA primers after amplification

Exonuclease I digests ssDNA in a 3′→5′ direction, but does not digest dsDNA. Although it requires the presence of magnesium and a free 3′-hydroxyl terminus for activity, it is active under a wide variety of buffer conditions and can be added directly into most reaction mixes. Exonuclease I can be heat-inactivated by incubation at 80°C for 15 minutes.

Unit Definition: One unit of Exonuclease I catalyzes the release of 10 nmol of acid-soluble nucleotides from heat-denatured calf thymus DNA in 30 minutes at 37°C under standard assay conditions.

Cat.# Concentration Quantity

Exonuclease I, E. coliX40501K 20 U/µl 1,000 Units X40505K 20 U/µl 5,000 Units X40520K 20 U/µl 20,000 Units

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rRNA Removal

RiboZero™ rRNA Removal KitsRemove >99% of the rRNA from a total RNA sample

The Ribo-Zero rRNA Removal Kits remove more ribosomal RNA (rRNA) from total RNA preparations than any other method. The kits are ideal for preparing RNA samples for whole-transcriptome RNA-Seq, random-primed cDNA synthesis, and other RNA analysis applications.

• Suitable for both intact and partially degraded RNA.• Recovers mRNA and small and large ncRNAs for whole-transcriptome RNA-Seq.• Single-pass, 90- to 120-minute process.• Recovers all fragments of nonribosomal RNAs in a partially degraded RNA sample.

Cat.# Quantity

Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat)RZH1046 6 ReactionsRZH110424 24 Reactions

Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) Low InputRZH1086 6 Reactions

Ribo-Zero™ rRNA Removal Kit (Meta-Bacteria)RZMB11086 6 Reactions

Ribo-Zero™ rRNA Removal Kit (Gram-Negative Bacteria)RZNB1056 6 Reactions

Ribo-Zero™ rRNA Removal Kit (Gram-Positive Bacteria)RZPB10106 6 Reactions

Ribo-Zero™ rRNA Removal Kit (Plant Leaf)RZPL11016 6 Reactions

Ribo-Zero™ rRNA Removal Kit (Plant Seed/Root) RZSR11036 6 Reactions

Total RNA samples were processed using the indicated method of rRNA removal or poly(A) enrichment. RNA-Seq libraries were prepared using the ScriptSeq™ Kit and sequenced on and Illumina® GAIIx sequencer. UHRR, Universal Human Reference RNA; BrRR, Brain Reference RNA; FFPE, human breast tumor.

Figure1.SummaryofRNA-SeqdataafterRibo-Zero™treatment.

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rRNA Removal

RiboZero™ Gold Kit (Human/Mouse/Rat)Remove cytoplasmic and mitochondrial rRNA

The Ribo-Zero Gold Kit (Human/Mouse/Rat) is an enhanced version of Epicentre’s popular Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat). The Ribo-Zero Gold Kit removes both cytoplasmic (nuclear-encoded) rRNA and mitochondrial rRNA from 1 to 5 µg of human, mouse, or rat total RNA preparations. The resulting rRNA-depleted RNA is suitable for RNA-Seq (Fig. 1), random-primed cDNA synthesis, and other RNA analysis applications.

• Suitable for both intact and partially degraded RNA.• Recovers mRNA and small and large ncRNAs for whole-transcriptome RNA-Seq.• Single-pass, 90- to 120-minute process.• Recovers all fragments of nonribosomal RNAs in a partially degraded RNA sample.

Cat.# Quantity

Ribo-Zero™ Gold Kit (Human/Mouse/Rat)RZHM11106 6 Reactions

Total RNA from MCF-7 cells was treated with either the standard Ribo-Zero Kit or the Ribo-Zero Gold Kit, and RNA-Seq libraries were prepared using the ScriptSeq Kit. Libraries were sequenced on Illumina® GAII and HiSeq 2000 sequencers. Data courtesy Vladimir Benes and Jonathon Blake, EMBL GeneCore, Heidelberg, Germany.

Figure1.ProfilesofRNA-seqlibrariespreparedaftertreatmentwiththeRibo-Zero™(A)andRibo-Zero™Gold(B)Kits.

28.10%

12.45%

31.68%

27.40%

0.02%0.05%

0.31%

24.04%

4.48%

32.05%

31.94%

6.72%

0.52% 0.24%

Aligning exomic

Partial exomic

Intronic

Genomic

16S mtrRNA

12S mtrRNA

28S rRNA

A B

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mRNA-Seq Sample Prep

Cat.# Quantity

ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina®-Compatible)SS10906 6 ReactionsSS10924 24 Reactions

RNA-Seq Barcode Primers (Illumina®-Compatible)RSBC10948 48 Reactions each Set of 12 Illumina®-compatible barcodes.

ScriptSeq™ mRNA-Seq Library Preparation KitsDirectional mRNA-Seq libraries in 3 hours

The ScriptSeq mRNA-Seq Library Preparation Kits* use a unique terminal-tagging technology* that simplifies the preparation of directional, paired-end libraries for Illumina® sequencing. The kits are suitable for any animal, plant, or bacterial species.

• Start with 50 ng or less of rRNA-depleted or poly(A)-enriched RNA.• Cluster-ready, directional libraries in about 3 hours without adaptor ligation.• Use Illumina-compatible barcodes (available separately) or user-defined barcodes.• Libraries demonstrate high concordance with MAQC qPCR data.• High percentage of mapped reads from intact, partially degraded, and FFPE RNA

samples.*Covered by issued and/or pending patents.

A. Intact UHRR and BrRR B. Fragmented UHRR and BrRR

Correlation between data obtained from ScriptSeq™ libraries and corresponding MAQC qPCR data. A) Libraries prepared from rRNA-depleted, intact UHRR and BrRR. B) Libraries prepared from partially fragmented, rRNA-depleted UHRR and BrRR. UHRR, Universal Human Reference RNA; BrRR, Brain Reference RNA.

Figure1.Correlationofgeneexpression.

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Small RNA-Seq Sample Prep

Cat.# Quantity

ScriptMiner™ Small RNA-Seq Library Preparation Kit (SinglePlex; Illumina®-Compatible)SMSP10908 8 ReactionsGenerates nonbarcoded, Illumina®-compatible libraries.

ScriptMiner™ Small RNA-Seq Library Preparation Kit (MultiPlex; Illumina®-Compatible)SMMP101212 12 Reactions Generates libraries containing Illumina®-compatible or user-defined barcodes. Illumina®-compatible barcodes are sold separately.

ScriptMiner™ Small RNA-Seq Library Preparation Kits (Illumina®-Compatible)Sequence the entire small-RNA transcriptome

The ScriptMiner Small RNA-Seq Library Preparation Kits (Illumina®-compatible) include Tobacco Acid Pyrophosphatase (TAP) to enable capture of the entire small-RNA transcriptome. Total RNA (1-5 µg) or size-selected RNA (100 pg) can be used as starting material.

• Single-day procedure.• Greatly reduced level of adaptor-dimer in the sequencing library. • Prepare libraries from miRNA and, optionally, from small 5′-capped and small

5′-triphosphorylated RNAs.• Strand-specific tagging of the RNA enables directional sequencing.

The optional TAP treatment in the ScriptMiner procedure enables the user to prepare libraries from small 5′-monophosphorylated RNAs, as well as small 5′-capped and small 5′-triphosphorylated RNAs that are not captured by conventional small RNA-seq library prep methods.

Figure1.AScriptMiner™librarycapturestheentiresmall-RNAtranscriptome.

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RNA-Seq

Tobacco Acid PyrophosphataseRemove the 5′ cap from mRNA for RNA-Seq

Tobacco Acid Pyrophosphatase (TAP) hydrolyzes the phosphoric acid anhydride bonds in the triphosphate bridge of the cap structure found in most eukaryotic mRNA, releasing the cap nucleoside and generating a 5′-monophosphorylated terminus on the RNA molecule. The resulting “decapped” 5′-monophosphorylated terminus may be ligated to a 3′-hydroxyl terminus using T4 RNA Ligase or dephosphorylated using APex™ Heat-Labile Alkaline Phosphatase. Similarly, TAP digests the triphosphate group at the 5′ end of prokaryotic transcripts, generating an RNA molecule with a 5′-monophosphorylated terminus.

Unit Definition: One unit of TAP releases 1 nmol of inorganic phosphate from m7GpppG in 30 minutes at 37°C under standard assay conditions.

Cat.# Concentration Quantity

Tobacco Acid Pyrophosphatase (TAP)T81050 5 U/µl 50 Units T19050 10 U/µl 50 Units T19100 10 U/µl 100 Units T19250 10 U/µl 250 Units T19500 10 U/µl 500 UnitsIncludes 10X Reaction Buffer.

Poly(A) Polymerase Tailing KitAdd poly(A) tails to RNA for RNA-Seq

Poly(A) Polymerase uses ATP as a substrate for template-independent addition of adenosine monophosphate to the 3′-hydroxyl termini of RNA molecules. The Poly(A) Polymerase Tailing Kit provides the enzyme and other reagents for quickly and easily adding a poly(A) tail to the 3′ end of any RNA.

Unit Definition: One unit of Poly(A) Polymerase catalyzes the incorporation of 1 nmol of AMP into acid-insoluble form in 10 minutes at 37°C under standard assay conditions.

Cat.# Concentration Quantity

Poly(A) Polymerase Tailing KitPAP5104H 50 Reactions 400 UnitsContents: Poly(A) Polymerase, 10X Reaction Buffer, 10 mM ATP, Sterile RNase-Free Water.

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RNA-Seq

APex™ Heat-Labile Alkaline PhosphataseDephosphorylate DNA or RNA for sequencing library prep

APex Heat-Labile Alkaline Phosphatase is an innovative enzyme preparation with improved performance over other available thermolabile alkaline phosphatases. APex Phosphatase removes the 5′ phosphate from all types of DNA ends, including 5′-protruding, blunt, and 5′-recessed ends, and from RNA ends. The enzyme is irreversibly heat-inactivated by incubation at 70°C for 5 minutes.

Unit Definition: One microliter of APex Heat-Labile Alkaline Phosphatase dephosphorylates 1 µg of pUC19 vector DNA digested with Hind III (5′-protruding ends), Hinc II (blunt ends), or Pst I (5′-recessed ends) in 10 minutes at 37°C.

Cat.# Concentration Quantity

APex™ Heat-Labile Alkaline PhosphataseAP49010 1 Reaction/µl 10 Reactions AP49050 1 Reaction/µl 50 Reactions AP49100 1 Reaction/µl 100 Reactions

RNA 5′ PolyphosphataseConvert 5’-triphosphorylated RNA to 5’-monophosphorylated RNA for RNA-Seq

RNA 5′ Polyphosphatase* is a Mg2+-independent phosphohydrolase discovered and characterized at Epicentre. The enzyme sequentially removes the γ and β phosphates from 5′-triphosphorylated RNA (such as primary RNA transcripts): 5′ pppN—OH 3′ → 5′ pN—OH 3′ + 2 Pi

RNAs with a 5′-diphosphorylated end are also converted to 5′-monophosphorylated RNA by RNA 5′ Polyphosphatase: 5′ ppN—OH 3′ → 5′ pN—OH 3′ + Pi

RNA Polyphosphatase has no activity on RNA with a 5′ cap (e.g., 5′ m7GpppN—OH 3′), or a 5′-monophosphorylated end (5′ pN—OH 3′). However, both NTPs and dNTPs are substrates for the enzyme, yielding the corresponding NMPs and dNMPs + inorganic phosphate: (d)NTP → (d)NMP + 2Pi

Unit Definition: One unit of RNA 5′ Polyphosphatase releases 1 nmol of inorganic phosphate from ATP in 1 hour at 37°C under standard assay conditions.*Covered by issued and/or pending patents.

Cat.# Concentration Quantity

RNA 5 PolyphosphataseRP8092H 20 U/µl 200 UnitsIncludes 10X Reaction Buffer.

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RNA-Seq

T4 RNA LigasePrepare 5’-tagged RNA for RNA-Seq

T4 RNA Ligase catalyzes the formation of a phosphodiester bond between a 5′-phosphoryl-terminated nucleic acid donor and a 3′-hydroxyl-terminated nucleic acid acceptor in a template-independent manner. The enzyme is ATP-dependent, and is active on a broad range of substrates including RNA, DNA, oligoribonucleotides, oligodeoxyribonucleotides, as well as numerous nucleotide derivatives.

Unit Definition: One unit of T4 RNA Ligase catalyzes the conversion of 1 nmol of 5′-phosphoryl termini in poly(A)18 into a phosphatase-resistant form in 30 minutes at 37°C under standard assay conditions.

Cat.# Concentration Quantity

T4 RNA Ligase   LR5010 5 U/µl 1,000 Units LR5025 5 U/µl 2,500 UnitsIncludes 10X Reaction Buffer and a 10-mM ATP Solution.

T4 RNA Ligase 2, Deletion MutantTag small RNAs for RNA-Seq

T4 RNA Ligase 2, Deletion Mutant, also known as T4Rnl2(1-249), is used to ligate single-stranded adenylated DNA or RNA oligonucleotides to small RNAs in the absence of ATP. The ligation products can be used for cloning or next-generation RNA sequencing. Performing the ligation reaction in the absence of ATP prevents circularization and other undesirable bimolecular reactions.

Unit Definition: One unit is the amount of enzyme required to give 50% ligation of a 22-mer RNA to the preadenylated end of a 17-mer DNA when both oligos are annealed to a complementary 39-mer DNA strand in 30 minutes at 37°C under standard assay conditions.

Cat.# Concentration Quantity

T4 RNA Ligase 2, Deletion MutantLR2D1132K 200 U/µl 2,000 UnitsLR2D11310K 200 U/µl 10,000 Units

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Illumina® BeadChip® Target Prep

Cat.# Quantity

TargetAmp™-Nano Target Labeling Kit for Illumina® Expression BeadChip®TAN07924 24 ReactionsTAN091096 96 ReactionsFor use with 25-500 ng of RNA

TargetAmp™-Pico Target Labeling Kit for Illumina® Expression BeadChip®TAP110610 10 ReactionsFor use with 50-500 pg of RNA

Additional TargetAmp kits are available. Visit www.epicentre.com for information.

Target RNA from the indicated source was labeled using either the TargetAmp-Nano or TargetAmp Pico kits. The target RNA was hybridized to either the Mouse Ref-8 Expression BeadChip or HumanHT-12 v4 Expression BeadChip (Illumina).

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

050 pg

Liver

50 pg

Muscle

100 pg

Liver

100 pg

Muscle

100 ng

Liver

100 ng

Muscle

500 ng

Ovary

500 ng

Testicle

Num

ber o

f gen

es d

etec

ted

RNA source and amount

TargetAmp™-Pico Kit

TargetAmp™-Nano Kit

TargetAmp™ Labeling Kits for Illumina® Expression BeadChip® ArraysLow-input RNA amplification and labeling

TargetAmp-NanoTargetLabelingKitforIlluminaExpressionBeadChip

• Produce microgram amounts of biotin-aRNA from 25 ng to 500 ng of RNA.• Perform the 6-hour TargetAmp-Nano Kit reaction and begin BeadChip hybridization

the same day.TargetAmp-PicoTargetLabelingKitforIlluminaExpressionBeadChip

• Produce microgram amounts of biotin-aRNA from 50 pg to 500 pg of RNA.• Ideal for very small samples, such as laser-capture microdissected (LCM) samples.

RNA source and amount

Num

ber o

f gen

es d

etec

ted

Figure1.GenesdetectedusingtargetlabeledwiththeTargetAmp™-NanoandTargetAmp™-Picokits.

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Cat.# Quantity

MessageBOOSTER™ cDNA Synthesis Kit from Cell Lysates KitMBCL90310 10 Reactions

MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit

Sensitive qRT-PCR from one cell…without purifying RNA!

A MessageBOOSTER cDNA Synthesis from Cell Lysates Kit reaction amplifies the poly(A) RNA directly from a cell lysate. and then converts the amplified RNA to cDNA that is ready for qPCR. There is no need to isolate total RNA.

• Low-abundance transcripts in a single cell are readily detected.• Hundreds of sensitive qRT-PCRs obtained from as little as a single cell. cDNA can be

archived for future use.• Linear RNA amplification process preserves the gene expression profile of the cell(s).

Rela

tive

Fluo

resc

ence

Uni

ts

Cycle

1:100

1:101:1,000

undiluted

Figure1.SensitivityoftheMessageBOOSTER™Kit.

qPCR was performed using undiluted (red), 1:10 diluted (green), 1:100 diluted (blue), and 1:1,000 diluted (purple) cDNA produced from a lysate of a single NRK cell. The low-abundance PBGD transcript was readily detected.

Single-Cell qRT-PCR Analysis

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Rapid Extraction of PCR-Ready DNA

Cat.# Quantity

QuickExtract™ DNA Extraction SolutionQE0905T 5 mlQE09050 50 ml

QuickExtract™ DNA Extraction KitsRapidly prepare PCR-ready DNA from various sample types

QuickExtract products offer a rapid and efficient method for extracting genomic DNA from virtually any sample for PCR-based assays. Most samples can be processed in 8 minutes with only two sequential heating steps.

• Fast—PCR-ready DNA in 3-8 minutes, ideal for high-throughput workflows.• Safe—No organic extraction.• Efficient—No columns, transfers, or sample loss.

M 1 2 3 4 5 6

M 1 2 3 4 5 6 7 M

Figure1.FailSafe™PCRamplificationofextractedDNA

Figure2.PCRamplificationofFFPEDNA

Buccal cells were extracted using the BuccalAmp™ DNA Extraction Kit, and all other samples with QuickExtract™ DNA Extraction Solution. PCR was performed using primers to amplify the regions indicated: Lanes 1-3, human β-globin (human buccal cells, HeLa cells, and human hair follicle, respectively); lane 4, transgenic mouse GAPDH (mouse tail snip); lane 5, E. coli 16S ribosomal RNA gene (bacteria); lane 6, transgenic SV40 T antigen (mouse tail snip).

DNA was extracted from a slide-mounted, FFPE-preserved human skeletal muscle tissue section with the QuickExtract™ FFPE DNA Extraction Kit. Two microliters of undiluted, extracted DNA was amplified with primers for three different loci: tumor protein 53 (TP53), dystrophin (DMD), and tumor necrosis factor (TNF). The products were separated on a 3% agarose gel and were visualized with SYBR® Gold. Lane M, 100-bp DNA ladder; lane 1, exon 2 of TP53; lane 2, exon 3 of TP53; lane 3, exon 11 of TP53; lane 4, exon 6 of DMD; lane 5, exon 50 of DMD; lane 6, exon 3 of DMD; lane 7, exon 4 of TNF.

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Products Time Applications SamplesTested

QuickExtract™ DNA Extraction Solution(QE0905T, QE09050)

3-8 min Genotyping, genetic studies, identity testing, viral/microbial screening

Hair follicles, feathers (quill-end cells), tissue-culture cells, buccal cells, zebrafish (organs, scales), mouse tail snips

BuccalAmp™ DNA Extraction Kits(BQ0901SCR, BQ0908SCR, BQ0916SCR)

3-8 min Genotyping, genetic studies, identity testing

Buccal cells from human or animal subjects. Catch-All™

Buccal Swabs included.

QuickExtract™ RNA Extraction Kit(QER09015, QER090150)

6 min RT-PCR Cultured cells: human, mouse, rat, E. coli, S. aureus

QuickExtract™ Plant DNA Extraction Solution(QEP80705, QEP70750)

8 min PCR, e.g., GMO testing Arabidopsis, barley, maize, emmer, pepper, rice, spelt, spinach, soybeans, wheat

QuickExtract™ Seed DNA Extraction Solution(QES08095T, QES080950)

8 min PCR, e.g., GMO testing Apple, cotton, sunflower, tomato, barley, maize, oats, rice, rye, wheat

QuickExtract™ Bacterial DNA Extraction Kit(QEB0905T, QEB09050)

15 min PCR, restriction digests, PFGE, optical mapping

Gram-positive: Bacillus subtilisBifidobacterium sppBrevibacterium linensClostridium perfringensLactobacillious plantarumListeria monocytogenesStaphylococcus equorumStreptococcus agalactiaeStreptococcus pyrogenesStreptococcus thermophilus

Gram-negative: E. coliSalmonella entericaSalmonella typhimuriumVibrio gazogenes

QuickExtract™ FFPE DNA Extraction Kit(QEF81805, QEF81050)

62 min Microsatellite, single-nucleotide polymorphisms (SNP), tumor heterogeneity studies, copy number variations (CNV), methylation analysis, short tandem repeats (STR)

Formalin-fixed, paraffin-embedded human tissue samples

QuickExtract™ FFPE RNA Extraction Kit(QFR82805, QFR82050)

32 min RT-PCR Formalin-fixed, paraffin-embedded human tissue samples

For more information, see: www.epicentre.com/quickextract

QuickExtract™ Family of Products

Rapid Extraction of Nucleic Acids

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Other Reagents

DNA and RNA Purification Cat.# Quantity

MasterPure™ Complete DNA and RNA Purification KitMC89010 5 DNA/10 RNA PurificationsMC85200 200 DNA/100 RNA Purifications

MasterPure™ DNA Purification Kit for Blood Version IIMB711740 For 40 ml of whole bloodMB711400 For 400 ml of whole blood

MasterPure™ RNA Purification KitMCR85102 100 Purifications

PCRCat.# Concentration Quantity

MasterAmp™ Extra-Long PCR KitMHF9220 50 Reactions

MasterAmp™ Extra-Long DNA Polymerase Mix (Enzyme Mix Only)QU92125 2.5 U/µl 125 UnitsQU92500 2.5 U/µl 500 Units QU9201K 2.5 U/µl 1,000 Units

Other EnzymesCat.# Concentration Quantity

RepliPHI™ Phi29 DNA Polymerase (enzyme only)PP031010 1 µg/µl (1,000 U/µl) 10 µg (10,000 Units)PP040110 0.1 µg/µl (100 U/µl) 10 µg (10,000 Units)

RepliPHI™ Phi29 Reagent SetRH031110 1 µg/µl (1,000 U/µl) 10 µg (10,000 Units)RH040210 0.1 µg/µl (100 U/µl) 10 µg (10,000 Units)Includes buffer, dNTPs, DTT, and enzyme at the indicated concentration/size.

Exo-Minus Klenow DNA PolymeraseKL0810250 5 U/µl 250 UnitsKL081001K 5 U/µl 1,000 UnitsKL080911K 10 U/µl 1,000 Units

Baseline-ZERO™ DNaseDB0711K 1 U/ul 1,000 MBUDB0715K 1 U/ul 5,000 MBU

RNase AMRNA092 5 mg/ml 2 ml

RiboShredder™ RNase BlendRS12100 1 U/µl 100 UnitsRS12500 1 U/µl 500 Units

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