Practical Hints for Successful PCR

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Transcript of Practical Hints for Successful PCR

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Effect of RNA integrity on real-time PCR results Tips to achieve a true RNA profiling suitable for real-time PCR studies

Dr. Ina Scheuerpflug, Ph.D.

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QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease.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.Legal disclaimerPractical Hints for RT-PCR2

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Critical steps for real-time PCR analysisRNA critical stepsPrinciples of RNA stabilizationCritical factors for RNA purificationChoosing the right RNA Purification methodmicroRNAs - micromanagers of gene expression

Improved methods for cDNA synthesis

Practical Hints for Real-Time PCR123

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DNADNAmRNATranscriptionNucleusCytoplasmtRNAsAttachedamino acidGrowingpeptidemRNAFunction and abundance of RNA

RNA speciesRelative abundancerRNA80-85%tRNAs, snRNAs (100 g)High RNA yield - excellent linearity down to 100 cells

Practical Hints for Real-Time PCR25

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gDNA contamination:

RNA conc.:

~150 ng /l lung~500 ng/l - kidney~900 ng/l - liver

RNeasy plus TissueExample: 10 mg rat tissue per prepUp to 1000x less gDNA, dep. on tissue

Practical Hints for Real-Time PCR26

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New RNeasy development: RNeasy Plus UniversalFor purification of total RNA from any type of tissue using gDNA Eliminator solutionOne-for-all tissue solutionTime-saving integration of RNeasy and QIAzol technologiesFast non-enzymatic removal of genomic DNAPure RNA for use in all downstream applications

Optimized protocols enable purification of high-quality RNA from any type of tissue, even difficult-to-lyse tissues. QIAzol Lysis Reagent included for lysing fatty tissue and other types of tissue, and RNeasy spin columns for purifying high-quality RNA. Kits are available in two formats: the RNeasy Plus Universal Mini Kit enables purification of RNA from up to 50 mg tissue and the RNeasy Plus Universal Midi Kit enables purification of RNA from up to 250 mg tissue. The RNeasy Plus Universal Mini Kit can be automated on the QIAcube.

Practical Hints for Real-Time PCR27

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Upgrade your expectations! RNeasy Plus UniversalQIAzol + gDNA Eliminator Solution + RNeasy Mini or Midi

Add gDNA Eliminator Solution and chloroform and shakeOne-for-all tissue kit with efficient gDNA Eliminator solutionPractical Hints for Real-Time PCR28

Sample to Insight28Lipid Kit procedure. QIAzol is used to homogenize the tissue prior to loading on the silica spin column.

Formaldehyde-fixed paraffin-embedded samples - ChallengesFormaldehyde fixation results in:

Protein-protein cross-linkingProtein-nucleic acid cross-linkingNucleic acid-nucleic acid cross-linking

Paraffin embedding means:

Extended incubation at >60C results in nucleic acid fragmentationDeparaffination is required prior to nucleic acid preparation

Harsh lysis conditions are required to break up tissueFragmentation of nucleic acidsRemaining formaldehyde modifications on nucleic acids interfere with enzymatic assays (reverse transcription, PCR)Practical Hints for Real-Time PCR29

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RNeasy FFPEOptimized deparaffination and lysisEnabling efficient RT-PCR by breaking formaldehyde crosslinksRNA preservation

gDNA eliminator spin columnEasy and efficient gDNA removal without compromising RNA yieldFaster and more economical than DNase digestion

Practical Hints for Real-Time PCR30

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Heat incubationHeat incubation in Prot.K digestion buffer helps to break formaldehyde crosslinks

shorter PK digest possible (15 min)better yieldbetter substrate for PCR

Limited by RNA stability: longer incubation / higher temp. will remove more of the formaldehyde modifications, but also results in more RNA fragmentation

(1 section, 10 m per prep; brain samples are from a different experiment)

Practical Hints for Real-Time PCR31

Sample to Insight31Data: RNA from rat liver FFPE sections (1 section, 10 m, per prep), SYBR green assays with 10 ng RNAVariability in yields is high due to sampling issues (cutting of sections)

Recovery of All Usable RNASmall RNA fragments efficiently recovered with RNeasy FFPE

Practical Hints for Real-Time PCR32RNA 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

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RNA from FFPE sections: quality / integrityQuality of RNA from FFPE samples is compromised due to:Fragmentation (heat + buffer conditions)

Formaldehyde crosslink / modification of RNA

For cDNA synthesis and PCR:avoid oligo-dT priming (better: random or gene-specific priming)choose short amplicons (500 nt are difficult to amplifyP, N positive, negative control RNA, 1-4 different heat incubation conditionsReg. Oligo-dT: distance of amplicon from poly-A tail is crucial. Also: formaldehyde x-link occurs preferentially on adenine residues!

Fast RNeasy FFPE ProcedureNo need for extra DNAse digestLysis with Prot. K digestion in only 15 min.In only 70 min all usable RNA.

80C, 15 min. to reverse formalin crosslinking14-30 l elution vol.

Practical Hints for Real-Time PCR34

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AllPrep solution: One Sample Two Analytes15-30 kb DNATotal RNA

Genomic DNA

RIN 10 RNAPractical Hints for Real-Time PCR35Total RNA and genomic DNA were purified from 10 mg samples of rat liver using the AllPrep DNA?RNA Mini Kit. A. Purified RNA samples were analyzed on a 1.2% formaldehyde agarose gel. B. Purified DNA samples were analyzed on a 0.8% agarose gel. M: markersTotal RNA was purified from 1 x 106 Jurkat cells using the AllPrep DNA/RNA Mini Kit and analyzed on the Agilent 2100 bioanalyzer. The RIN value was a maximum of 10, indicating intact RNA. M: markers; 18S: 18S rRNA; 28S: 28S rRNA

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Short Streamlined Procedure for DNA/RNA Purification Novel AllPrep DNA columnBind Wash - EluteDNA and RNA in 35 minutesRNeasy column

123Practical Hints for Real-Time PCR36

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Critical steps for real-time PCR analysisRNA critical stepsPrinciples of RNA stabilizationCritical factors for RNA purificationChoosing the right RNA Purification methodmicroRNAs - micromanagers of gene expression

Improved methods for cDNA synthesis

Practical Hints for Real-Time PCR1237

Sample to Insight37

microRNAs - micromanagers of gene expressionCharacteristic of microRNAsNaturally occurring, endogenous small RNAA mature miRNA is approximately 22 nt longRegulate at least 1/3 of the protein encoding genes> 500 microRNAs in humanMediate Post Transcriptional Gene Silencing either by translational repression or target mRNA degradationA typical human cell harbors 1.000 to 200.000 miRNAs in patterns unique to particular cell typesOne miRNA might bind 100 or more target mRNAsA single mRNA might have target sites for several different miRNAshttp://microrna.sanger.ac.uk

Fine-tuning of gene expression Cell fateDifferentiationMorphogenesisDevelopmentMany aspects of physiologyPractical Hints for Real-Time PCR38

Sample to Insight38Regulate at least 1/3 of the protein encoding genes ( Lewis at al., 2005)Regulate at least 1/3 of the protein encoding genes ( Lewis at al., 2005)

Biogenesis of microRNA

Transcribed by RNA Polymerase II as - pri-miRNAs

In the nucleus, Pri-miRNAs are processed to ~ 70 nt hairpin-like pre-miRNAs by Drosha

Pre-miRNAs are then exported by Exportin 5

In the cytosol pre-miRNAs are processed to mature miRNAs by Dicer

These miRNAs are incorporated into the RISC (RNA-Induced Silencing Complex)

miRNAs with imperfect base pairing to the target mRNAlead to translational repression and/or mRNA degradation

CytosolPractical Hints for Real-Time PCR39

Sample to Insight39The miRNA pathway is closely related to the siRNA pathway. A crucial difference is that siRNA is an exogenous system, in which double-stranded RNA enters the cell from an external source, such as from a viral infection.

In contrast, the miRNA system is endogenous; RNAs are transcribed from genomic DNA in the nucleus, and mature miRNAs regulate endogenous genes.During RNAi, siRNAs mediate degradation of target mRNAs with perfect complementarily resulting in gene silencing. miRNAs are often partially complementary to target mRNAs and are thought to down regulate gene expression by preventing translation.

miRNAs are transcribed by RNA Polymerase II as long primary transcripts (pri-miRNAs), which may contain more than one miRNA. The nuclear RNAse III like enzyme Drosha processes these pri-miRNAs into ~70 nt stem loop containing pre-miRNAs. Pre-miRNAs are exported into cytoplasm by Exportin 5/RanGTP and then processed further into mature miRNAs by cytoplasmic enzyme Dicer. both siRNAs & miRNAs are the key effectors of this RISC mediated PTGS mechanism. Researchers have been using a synthetic dsRNA (siRNA) as a tool to understand the gene function, by taking advantage of the naturally occurring PTGS Pathway. siRNAs target the cleavage destruction of perfectly (or nearly perfect) base paired cognate mRNA, while miRNAs w