Critical Steps for Real-Time PCR Analysis: Tips and Solutions to Achieve Efficient and Precise Gene...

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Critical Steps for Real-Time PCR AnalysisTips and solutions to achieve efficient and precise gene expression results

Lily Xuanyan Xu – Global Market Manager QIAGEN

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Legal disclaimer

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.

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Agenda

Gene expression workflows

1.1 Advantages of real-time PCR

Critical steps for real-time PCR analysis

2.1 Stabilization of RNA in-sample

2.2 RNA isolation

2.3 cDNA synthesis

QIAGEN gene expression workflow solutions

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2.2 RNA isolation

2.3 cDNA synthesis


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Gene expression profiling

Understanding biological pathways and cellular systemGene expression is regulated at different levels• Transcriptional

• Post-transcriptional

• Translational

• Post-translational

Available experiment methods• Real-time PCR

• Northern blotting

• DNA microarray

• Next-Gen sequencing

Overview of gene expression profiling

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Gene expression profiling

Understanding biological pathways and cellular system.Gene expression is regulated at different levels:• Transcriptional

• Post-transcriptional

• Translational

• Post-translational

Available experiment methods:• Real-time PCR

◦ Sensitive

◦ Quantitative

◦ Reliable

• Northern blotting

• DNA microarray

• Next-Gen sequencing

Overview of gene expression profiling

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Basic gene expression workflow using real-time PCR

RNA quality (purity and integrity) plays a critical role in the accuracy, reproducibility and relevance of downstream analysis.

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Challenges in gene expression workflows

• DNase contamination

• Salt carry-over

• Phenol carry-over

• gDNA contamination

• Complex samples

• Low yield

• Extraction techniques

• RNase contamination

• Improper storage

• RNA degradation

• Sample handling

• Low abundance transcripts

• Large transcripts

• High GC content

• Secondary structure

• Introducing bias

• Low yield

• gDNA removal

• IC controls

• Pipetting errors

• Room temperature setup

• Primer design

• Sensitivity

• Restrictions due to the cyclers

• Calibration and maintenance efforts

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Agenda





2.2 RNA isolation

2.3 cDNA synthesis


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2.2 RNA isolation

2.3 cDNA synthesis


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Sample collection and stabilization

Changes in mRNA levels following sample harvesting

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Effect of stabilization

Purification of RNA without degradation

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Principles of RNA stabilization

What do we want to achieve?

• Prevent induction of new RNA transcription

• Avoid regulated turnover of mRNA

• Impede unspecific degradation of RNA – either enzymatically (due to release of nucleases

from specific cell types or compartments) or chemically (pH, temperature, etc. dependent)

Approaches:

• Agents that bind directly to nucleic acids

• Agents that lyse cells

• Agents that inhibit, denature and / or precipitate nucleases (and proteins in general)

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Requirements for stabilization of blood and tissue samples

Blood contains only individual cells but has a high protein content:• Diffusion rates of the stabilizing agent are not problematic

• Agents that precipitate or cross-link proteins are detrimental

• In principle, blood volume is not limited (container is limited)

• One-step stabilization technology is required

Tissue contains connected cells:• Diffusion rates of the stabilizing agent are critical

• Thickness of the tissue sample is limited

• Multi-step technology applicable as tissue pieces are transferrable

• Protein precipitation or cross-linking is not an issue

14Practical Hints for Real-Time PCR

QIAGEN offers a variety of stabilization reagents for several different sample types: blood, tissue, cells, bone marrow, bacteria, saliva etc.

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Agenda





2.2 RNA isolation

2.3 cDNA synthesis


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2.2 RNA isolation

2.3 cDNA synthesis


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Sample disruption and homogenization

Disruption and homogenization: two distinct steps.Disruption: complete disruption of tissue structure, cell walls and plasma membranes of cells is required to release all the RNA contained in the sample. • Different samples require different methods to achieve complete disruption.

• Incomplete disruption results in significantly reduced yields.

Homogenization: Homogenization is necessary to reduce the viscosity of the cell lysates produced by disruption. Homogenization shears the high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. • Incomplete homogenization results in inefficient binding of RNA and therefore significantly

reduced yields.

Efficient disruption and homogenization - an absolute requirement

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Manual disruption and homogenization

Classic Method

Step 1: Freeze the sample in liquid nitrogen

Step 2: Grind tissue into a powder using a mortar and pestle• Approach works well but it is not consistent

Step 3: Resuspend powdered sample in chaotropic lysis buffer• Genomic DNA is still high molecular weight and will add viscosity to

the sample that can clog spin filters

Step 4: Shear with needle and syringe, improving the efficiency of gDNA removal from columns• Care needed to prevent foaming but still be effective

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Simultaneous Disruption and Homogenization

TissueRuptor• One sample / run

• Disposable probes

• Rotor-stator

TissueLyser LT• Up to 12 samples in parallel

• Bead mill

TissueLyser II• Up to 48 or 192 samples

in parallel

• Bead mill

Mechanical disruption and homogenization

Human / animal tissuePlant tissue

Human / animal tissuePlant tissueBacteriaYeast

Human / animal tissuePlant tissueBacteriaYeast

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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).

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Comparison of RNA yields with different homogenization methods

QIAshredder – fast and simple homogenization of cell lysates

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mRNA enrichment

mRNA enrichment

Selective exclusion of RNA < 200 nt

mRNA

rRNA > 200 nt

tRNA, snRNA, miRNA < 200 nt

Practical Hints for Real-Time PCR 22

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Choosing the right RNA purification product / protocol

Is the RNA purification procedure appropriate for the amount of starting material?• Overloading usually results in lower purity, lower yields

• With small samples, it is important to use an efficient RNA purification method

RNA purification from large samples: • Provides a pool of RNA that can be used for multiple experiments

• May be required to get sufficient amounts of RNA from samples that have low RNA content

• High binding capacity – usually also implies higher dead volume


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Special consideration: Difficult-to-lyse tissues

Cells

Yeast

Some tissues• Liver• Spleen• Kidney

Lysis: Phenol-free lysis buffer

Fatty Tissues• Brain• Skin • Adipose tissues – e.g. breast

Lysis: Phenol / guanidine reagent

Fibrous Tissues• Muscle• Heart• Trachea• Lung

Lysis: Proteinase K or phenol / guanidine reagent

Easy to lyse Difficult to lyse

Some tissues require stronger lysis conditions

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Special consideration: Fibrous tissue

Isolating RNA from heart, muscle and other fibrous tissue:

• Contractile proteins, connective tissue and collagen, which can all interfere with the isolation process

• Sample needs to be treated with a protease or phenol containing lysis reagents

• Proper conditions that do not degrade RNA, such as with an RNase-free proteinase K digest (RNeasy Fibrous Tissue Kit)

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Precipitation vs. spin columns

Precipitation Spin columns

Cheap, no kits required, scalable Higher material cost

More handling, longer incubation times Fast, easy to use

Often several rounds of precipitation required for decent purity High purity

Risk to lose RNA pellet, especially with small samples

Special formats for different sample types and sizes, including very small samples

More room for error; inaccurate pipetting between phases can cause phenol carryover

Consistent, no phenol carry-over

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Special consideration: FFPE samples

• Remove and fix tissue as quickly as possible• Use tissue samples no more than 5 mm thick and do

not over-fix (max 24 hours)• Use high-quality reagents for paraffin embedding,

without additives• Avoid sample staining, if possible• Store FFPE samples appropriately

Note: RNA remains intact for up to a year when stored at 4ºC

• Use an appropriate deparaffinization step• Have a crosslink-reversal step during RNA isolation

Minimize the effects of FFPE storage on RNA transcripts

Complete FFPE guide: Critical factors for molecular analysis of FFPE samples

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FFPE sample – challenges

Formaldehyde fixation results in:• Protein-protein cross-linking• Protein-nucleic acid cross-linking• Nucleic acid-nucleic acid cross-linking

Paraffin embedding means:• Extended incubation at >60°C results in nucleic acid fragmentation• Deparaffination is required prior to nucleic acid preparation

• Harsh lysis conditions are required to break up tissue• Fragmentation of nucleic acids• Remaining formaldehyde modifications on nucleic acids interfere with enzymatic assays

(reverse transcription, PCR)


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Heat incubation

Heat incubation in proteinase K digestion buffer helps to break formaldehyde crosslinks• Shorter PK digest possible (15 min)

• Better yield

• Better substrate for PCR

Limited by RNA stability: Longer incubation / higher temperature will remove more formaldehyde modifications, but will also result in more RNA fragmentation.

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


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Recovery of all usable RNA

Small RNA fragments efficiently recovered with RNeasy FFPE


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

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RNA from FFPE sections: quality / integrity

Quality 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, if possible)

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Special consideration: RNA from human blood samples

• Very small amounts of RNA

• RNA integrity – presence of RNases

• Cellular RNA or exosomal RNA

• Contaminants must be removed – anticoagulants heparin and EDTA, and naturally occurring enzyme inhibitors, all of which can interfere with downstream RNA analysis

PaxGene webinar and exosomes webinar available

Some challenges of human blood samples:

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Special consideration: Plant material

Things to consider:

• Plant metabolites are difficult to remove

• Healthy young tissues recommendedo RNA yields often higher since young

tissue generally contains more cells and fewer metabolites than the same amount of older tissue

• Many “home-made” protocols RNA isolation recommend growing plants in darkness for 1 to 2 days before harvesting to prevent high levels of plant metabolite accumulation

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Dedicated kits could solve the problem

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Special consideration: RNA from bacteria and viruses

Bacterial RNA

• Bacterial mRNA has no 5’ cap and rarely has a poly-A tail

• mRNA isolation by hybrid capture is impossible

• The RNeasy Protect Bacteria Kit is makes bacterial gene expression studies possible

Viral RNA 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

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Back to Basics: RNA Isolation 37

Removal of genomic DNA contamination

Why is removing gDNA so important?

Trace amounts of gDNA 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.

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Removal of genomic DNA contamination

Eliminate genomic DNA contamination either during RNA purification or just prior to cDNA synthesis.

DNase digestion:• On-column, during isolation (DNA already bound to column)

• After RNA isolation, prior to cDNA synthesis

Non-enzymatic removal (columns or reagents).

Design primers to avoid coamplification of DNA targets.

QIAGEN solutions:• DNase digestion (with or without DNase Booster)• gDNA Eliminator Column – RNeasy Plus Kits• gDNA Eliminator Solution – RNeasy Plus Universal Kit

o The gDNA Eliminator Solution is a novel, non-enzymatic solution that reduces gDNA contamination of the aqueous phase. It does not contain DNase

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microRNAs - micromanagers of gene expression

Characteristic of microRNAs:• Naturally occurring, endogenous small RNA

• A mature miRNA is approximately 22 nt long

• Regulate at least 1/3 of the protein encoding genes

• > 500 microRNAs in human

• Mediate post-transcriptional gene silencing either by translational repression or target mRNA degradation

• A typical human cell harbors 1,000–200,000 miRNAs in patterns unique to particular cell types

• One miRNA might bind 100 or more target mRNAs

• A single mRNA might have target sites for several different miRNAs

• http://microrna.sanger.ac.uk

Fine-tuning of gene expression:• Cell fate

• Differentiation

• Morphogenesis

• Development

• Many aspects of physiology


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Biogenesis of microRNA

1. Transcribed by RNA polymerase II as pri-miRNAs.

2. In the nucleus, pri-miRNAs are processed to ~70 nt hairpin-like pre-miRNAs by DROSHA.

3. Pre-miRNAs are then exported from the nucleus by Exportin 5.

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

5. These miRNAs are incorporated into the RNA-induced silencing complex (RISC).

6. miRNAs with imperfect base pairing to the target mRNA• Lead to translational repression and/or mRNA

degradation

Cytosol


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RIBOSOME

miRNA – how it works

3`5` AAAAAmRNA

RISC-like complex 5`P3ÒH

5`P3ÒH

3ÙTR3ÙTR Seed Region match

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miRNA is incorporated in RISC and binds to a 7–9 nucleotide region in the 3úntranslated region of the mRNA (= 3ÚTR seed region).This binding prevents the ribosome from translation of the GOI.→ The protein is down regulated.


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miRNAs will likely enter the clinic as biomarkers and diagnostics

Most applications are expected to be in oncology because many miRNAs affect the cell cycle.

• Half of all human miRNAs discovered so far are expressed abnormally in at least one cancer

• Some miRNA profiles are specific to one cancer, others are common to many cancers

Regardless of whether the change in expression is causal or a downstream effect, miRNA profiling is helping discover new

markers for human disease classification.


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miRNA isolation technologies

Cells / Tissue – miRNeasy MiniFNB – miRNeasy MicroFFPE – miRNeasy FFPEBlood – miRNeasy Serum/PlasmaSerum / Plasma – miRNeasy Serum /

Plasma

miRNeasy – efficient purification of miRNA from different starting materials

Highly pure RNA without phenol carryover

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Efficient copurification from wide array of tissues


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2.2 RNA isolation

2.3 cDNA synthesis


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2.2 RNA isolation

2.3 cDNA synthesis


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Critical factors for an efficient reverse trancription step

Reverse transcription enzyme efficiency is influenced by:

• Quality / quantity of the RNA starting materialo Phenol contaminationo Alcohol contaminationo Salt contaminationo Protein inhibitors

• Affinity to the RNA to avoid secondary structure effects• Choice of priming method

o Random priming vs. oligo-dT priming vs. gene-specific priming

• Genomic DNA contamination• Conditions for one-step vs. two-step RT-PCR• Sequences near the 5‘ end affect the cDNA yield• Use of internal controls is critical

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Effects of complex secondary structure on RT-PCR

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Eliminating genomic DNA contamination

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Improved methods for cDNA synthesis

• QuantiNova Reverse Transcriptase◦ Highly efficient transcription for sensitive detection even of low abundance targets◦ Use of a wide range of RNA amounts (10 pg–5 μg)◦ High affinity for RNA – leads to high sensitivity◦ Up-scaling option for larger input volumes (up to double volume) ◦ Successful use even for difficult templates◦ Plus RNase inhibitor

• gDNA removal buffer◦ Efficient removal of gDNA (> 1000 fold reduction) is integrated in the protocol

• RT-Primer Mix◦ Optimized mix of oligo-dT and random primers

• Internal control◦ Optional use of internal control included to monitor cDNA synthesis efficiency

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QuantiNova Reverse Transcription Kit – protocol

• With unique Internal Control RNA• With gDNA removal

Fast and convenient protocol:1. RNA (potentially contaminated with gDNA)

◦ Add gDNA Removal Buffer (incl. RNase Inhibitor)◦ Optionally spike in Internal Control RNA◦ 2 min 45°C

2. Synthesize cDNA◦ Add 5 µl RT-Master Mix ◦ 3 min 25°C, 10 min 45°C, 5 min 85°C cDNA (incl. IC cDNA, if

applicable) ◦ Stable storable, colorable with QN Yellow Template Dye

3. Stop reaction (95°C, 3 min) get cDNA without gDNA contamination!!!

cDNA synthesis procedure in only 20 minutes


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QuantiNova Reverse Transcription Kit – Internal Control

QuantiNova IC reliably indicates inhibition or failure of RT or qPCR reaction.

It is a synthetic RNA that can be optionally used to monitor succesful RT.

Different amounts of SDS spiked into the PCR reaction. The IC indicated inhibition by delayed CT values.Reliable in-process monitoring of RT and qPCR performance.


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QuantiNova Reverse Transcription Kit – gDNA removal

Efficient, optional gDNA removal prevents CT shifts caused by DNA contamination.

Precise mRNA quantification even if exon spanning primers cannot be used

10 ng gDNA in PCR reaction as positive control

100 & 10 ng gDNA spiked in without gDNA removal in RT-rxn

NTC

100 & 10 ng gDNA spiked in with gDNA removal


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Effect of primer choice

Amplicon – 3‘ end: 2 kb

(mix or oligo-dT)

(N)x Oligo-dT+(N)x

Oligo-dT

Amplicon – 3‘ end: 6 kb

(mix or oligo-dT)

Oligo-dT

Oligo-dT+(N)x

(N)x

^ 10 kb transcript (amplicon 2kb and 6kb away from 3´ end)

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• High sensitivity due to novel enzyme

• Synthesize cDNA and remove genomic DNA in 20 minutes

• Integrated genomic DNA removal step

• Reverse transcription from all regions due to primer mix (5’ region)

• Internal control

Improved methods for cDNA synthesis

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Agenda





2.2 RNA isolation

2.3 cDNA synthesis


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2.2 RNA isolation

2.3 cDNA synthesis


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Challenges in gene expression workflows

• DNase contamination

• Salt carry-over

• Phenol carry-over

• gDNA contamination

• Complex samples

• Low yield

• Extraction techniques

• RNase contamination

• Improper storage

• RNA degradation

• Sample handling

• Low abundance transcripts

• Large transcripts

• High GC content

• Secondary structure

• Introducing bias

• Low yield

• gDNA removal

• IC controls

• Pipetting errors

• Room temperature setup

• Primer design

• Sensitivity

• Restrictions due to the cyclers

• Calibration and maintenance efforts

RNA quality (purity and integrity) plays a critical role in the accuracy, reproducibility and relevance of downstream analysis.

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QIAGEN solutions

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Useful resources

• Gene expression workflow solutions: www.qiagen.com/gene-expression-workflow

• Review: RNA integrity and the effect on the real-time qRT-PCR performanceFleige S. Phaffl MW. Mol Aspects Med. 2006 Apr-Jun;27(2-3):126-39. Epub 2006 Feb 15. DOI: 10.1016/j.mam.2005.12.003

• RNA resource centre: https://www.qiagen.com/qdm/rna/resources

• Blogs on PCR solutions:http://biomarkerinsights.qiagen.com/category/pcr-solutions/

• Real-time PCR and RT-PCR on GeneQuantification.info:http://www.gene-quantification.com/real-time.html

• Troubleshooting guide:https://www.qiagen.com/support/troubleshooting/

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2.2 RNA isolation

2.3 cDNA synthesis


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2.2 RNA isolation

2.3 cDNA synthesis


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Thank you for attending today’s webinar!

Contact QIAGEN Technical ServiceCall: 1-800-426-8157 for US

Call: +49 2103-29-12400 for EU

Email: [email protected]@[email protected]

Lily Xu Xuanyan, [email protected]

Questions?

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Critical Steps for Real-Time PCR Analysis: Tips and Solutions to Achieve Efficient and Precise Gene...

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