RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
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Back to basics: Fundamental Concepts and Special Considerations in RNA Isolation
1. Sample to Insight
Back to basics: Fundamental concepts and special considerations
in RNA isolation
Abhishek Sharma, M.Sc.
Senior Global Market Manager, Sample Technologies
RNA
Back to basics: Fundamental concepts and special considerations in RNA isolation 1
2. Sample to Insight
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.
Back to basics: Fundamental concepts and special considerations in RNA isolation 2
3. Sample to Insight
Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 3
4. Sample to Insight
Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 4
5. Sample to Insight
RNA universe
Name: Ribonucleic acid
Abbreviation: RNA
Destination: Throughout the cell (nucleus, nucleolus,
cytoplasm, ribosomes, mitochondria, chloroplasts,
and endoplasmic reticulum) and in exosomes for
communication between cells
Types:
⢠Messenger RNA (mRNA), transcribed from DNA, serves as
a template for synthesis of proteins
⢠Ribosomal RNA (rRNA) present in ribosomes for protein
synthesis
⢠Transfer RNA (tRNA) deliver amino acids to the ribosomes
for protein synthesis
⢠miRNAs are noncoding RNAs that mediate post-
transcriptional gene regulation
⢠Long non-coding RNAs (lncRNA) range from 200
nucleotides to100 kb in length with diverse functions
including epigenetics, alternative splicing and nuclear
import.
To get the complete picture:
⢠Download our RNA Universe App:
www.qiagen.com/qdm/app/universe/login.aspx
Did you know?
Thirty
scientists have
received Nobel Prizes
for research on
RNA
Back to basics: Fundamental concepts and special considerations in RNA isolation 5
6. Sample to Insight
RNA distribution in cells
RNA content of a typical human cell
Parameter Amount
Total RNA per cell <1â30 pg
Proportion of total RNA in nucleus ~14%
DNA:RNA in nucleus ~2:1
mRNA molecules 2 x 105 â 1 x 106
Typical mRNA size 1900 nt
RNA distribution in a typical mammalian cell
RNA species Relative amount
rRNA (28S, 18S, 5S) 80â85%
tRNAs, snRNAs, low MW species 15â20%
mRNAs 1â5%
mRNA classification based on abundance
Abundance Copies/cell No. of different messages/cell Abundance of each message
Low 5â15 11,000 <0.004%
Intermediate 200â400 500 <0.1%
High 12,000 <10 3%
Back to basics: Fundamental concepts and special considerations in RNA isolation 6
7. Sample to Insight
Generic gene expression workflow
Back to basics: Fundamental concepts and special considerations in RNA isolation 7
Sample
collection
Disruption Purification
cDNA
synthesis
Analysis
8. Sample to Insight
Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 8
9. Sample to Insight
Handling RNA â The key factors
Working quickly but carefully is key!
General handling
⢠Always wear latex or vinyl gloves to prevent RNase
contamination
⢠Change gloves frequently and keep tubes closed
whenever possible
Disposable plasticware
⢠The use of sterile, disposable polypropylene tubes is
recommended
⢠These tubes are generally RNase-free and do not
require pretreatment to inactivate RNases
Non-disposable plasticware
⢠Non-disposable plasticware should be treated before
use to ensure that it is RNase-free
⢠Rinse thoroughly with 0.1 M NaOH, 1 mM EDTA
followed by RNase-free water
⢠Alternatively, chloroform-resistant plasticware can be
rinsed with chloroform to inactivate RNases
Back to basics: Fundamental concepts and special considerations in RNA isolation 9
10. Sample to Insight
Handling RNA â The key factors
Glassware
⢠Glassware should be cleaned with a detergent,
thoroughly rinsed, and oven baked at 240°C for at least
4 hours before use
⢠Autoclaving alone will not fully inactivate many RNases
⢠An alternative is DEPC treatment: Fill glassware with
0.1% DEPC (0.1% in water), incubate overnight (12
hours) at 37°C, and then autoclave or heat to 100°C for
15 min
Electrophoresis tanks
⢠Electrophoresis tanks should be cleaned with detergent
solution (e.g., 0.5% SDS), thoroughly rinsed with
RNase-free water, and then rinsed with ethanol and
allowed to dry
⢠Plastics used for some electrophoresis tanks are not
resistant to ethanol
For complete tips, go to www.qiagen.com/qdm/rna/resources/
Working quickly but carefully is key!
Back to basics: Fundamental concepts and special considerations in RNA isolation 10
11. Sample to Insight
Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 11
12. Sample to Insight
Generic gene expression workflow
Back to basics: Fundamental concepts and special considerations in RNA isolation 12
Sample
collection
Disruption Purification
cDNA
synthesis
Analysis
13. Sample to Insight
Sample collection and stabilization
Back to basics: Fundamental concepts and special considerations in RNA isolation 13
Changes in mRNA levels following sample harvesting
mRNA
level
Sample harvest and processing
14. Sample to Insight
RNAlater RNA Stabilization Reagent
Purification of RNA without degradation
Back to basics: Fundamental concepts and special considerations in RNA isolation 14
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Immediate RNA stabilization at room temperature
Storage and transport of samples at ambient temperature
RNAprotect Reagent
⢠Cells
⢠Bacteria
⢠Saliva
RNAlater RNA Stabilization Reagent
⢠Freshly harvested tissue
Sample RNA stabilization RNA stabilization and purification
Tissues RNAlaterÂŽ RNA Stabilization Reagent
RNAlater TissueProtect Tubes
RNeasyÂŽ Protect Kits
Mammalian cells RNAprotectÂŽ Cell Reagent RNeasy Protect Cell Mini Kit
Human saliva RNAprotect Saliva Reagent RNeasy Protect Saliva Mini Kit
Human blood PAXgene⢠Blood RNA Tubes PAXgene Blood RNA Kit
Bacteria RNAprotect Bacteria Reagent RNeasy Protect Bacteria Kits
Back to basics: Fundamental concepts and special considerations in RNA isolation 15
16. Sample to Insight
Generic gene expression workflow
Sample
collection
Disruption Purification
cDNA
synthesis
Analysis
Back to basics: Fundamental concepts and special considerations in RNA isolation 16
17. Sample to Insight
Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
Step 1: Sample disruption and homogenization4.1
Step 2: Isolating RNA from different samples â tips and tricks4.2
Step 3: gDNA removal for sensitive applications4.3
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 17
18. Sample to Insight
Sample disruption and homogenization
Disruption and homogenization are 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 is an absolute requirement.
Back to basics: Fundamental concepts and special considerations in RNA isolation 18
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Manual disruption using a mortar and pestle
Classical method
Using liquid nitrogen to freeze the sample
Mortar and pestle to grind the tissue to a powder
Powdered sample needs to be resuspended in a
chaotropic lysis buffer
Shear the genomic DNA with a needle and
syringe which improves the efficiency of
removing the genomic DNA from the columns
Approach works well
but is incomplete
Genomic DNA is high in
molecular weight which
adds viscosity to the
sample clogging
spin filters
Back to basics: Fundamental concepts and special considerations in RNA isolation 19
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Comparison of RNA yields with different homogenization methods
QIAshredder â fast and simple homogenization of cell lysates
Back to basics: Fundamental concepts and special considerations in RNA isolation 20
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Systems for mechanical disruption and homogenization
Human/animal tissue
Plant tissue
Human/animal tissue
Plant tissue
Bacteria
Yeast
Human/animal tissue
Plant tissue
Bacteria
Yeast
Replacing methods such as manual disruption using a mortar and pestle
Up to 48 or 192 samples/run
TissueLyser II
1 sample/run
TissueRuptor
Up to 12 samples/run
TissueLyser LT
Back to basics: Fundamental concepts and special considerations in RNA isolation 21
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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)
Back to basics: Fundamental concepts and special considerations in RNA isolation 22
RNA yields
RNA yield
(ng/Âľl)
23. Sample to Insight
Generic gene expression workflow
Sample
collection
Disruption Purification
cDNA
synthesis
Analysis
Back to basics: Fundamental concepts and special considerations in RNA isolation 23
24. Sample to Insight
Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
Step 1: Sample disruption and homogenization4.1
Step 2: Isolating RNA from different samples â tips and tricks4.2
Step 3: gDNA removal for sensitive applications4.3
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 24
25. Sample to Insight
Precipitation vs. spin columns
Precipitation Spin columns
Cheap, no kits required, scalable Higher material cost
Longer handling and incubation times Fast, easy to use
Several rounds of precipitation are
often 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
Back to basics: Fundamental concepts and special considerations in RNA isolation
26. Sample to Insight
Special considerations for various sample types
Isolating RNA from heart, muscle and other fibrous tissues
⢠Contractile proteins, connective tissue and collagen can interfere with the isolation process
⢠Sample needs to be treated with a protease or phenol containing lysis reagents
⢠Maintain proper conditions that do not degrade RNA, e.g., use RNase-free proteinase K
⢠RNA was isolated using the RNeasy Fibrous Tissue Mini Kit
⢠Real-time, quantitative RT-PCR was performed with 100 ng total RNA using the QuantiTect
Probe RT-PCR Kit and primers and probes specific for alpha-actin
Back to basics: Fundamental concepts and special considerations in RNA isolation 26
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RNA from FFPE samples
Minimize the effects of FFPE storage on RNA transcripts
⢠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
⢠RNA remains intact for up to 1 year when
stored at 4ÂşC
⢠Use an appropriate deparaffinization step
⢠Have a crosslink reversal step during RNA
isolation
The complete FFPE guide: Critical factors for molecular analysis of FFPE samples
Back to basics: Fundamental concepts and special considerations in RNA isolation 27
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miRNeasy FFPE Kit
Successful detection of miRNA and mRNA from the same eluate
⢠Rat liver tissue was formalin fixed for 24 hours or 60 hours, followed by purification of total
RNA including miRNA
⢠Purified RNA was used as a template in quantitative, real-time RT-PCR using the miScript
PCR System
Back to basics: Fundamental concepts and special considerations in RNA isolation 28
CT value
29. Sample to Insight
Isolating RNA from human blood samples
⢠Very small amounts of RNA
⢠RNA integrity - Presence of RNases
⢠Cellular RNA or exosomal RNA
⢠Contaminants interfere with downstream
RNA analysis
⢠Contaminants include anticoagulants â
heparin and EDTA, and naturally
occurring enzyme inhibitors
Back to basics: Fundamental concepts and special considerations in RNA isolation 29
30. Sample to Insight
Isolating RNA from plant samples
⢠Several plant metabolites â difficult to
remove
⢠As a general guideline, healthy, young
tissues are recommended
⌠Young tissues yield more RNA compared to
older ones as they contain more cells and
fewer metabolites
⢠Many âhome-madeâ protocols for RNA
isolation recommend growing plants in
darkness for 1 to 2 days before harvesting
to prevent high levels of plant metabolite
accumulation
Back to basics: Fundamental concepts and special considerations in RNA isolation 30
31. Sample to Insight
Dedicated kits could solve the problems with RNA isolation
Reproducible purification of intact RNA from plant tissues â the RNeasy Plant Mini Kit
Tomato Arabidopsis Cotton Maize Canola
RNA isolated from the leaves of five plant species
⢠Frozen plant leaves were disrupted using the TissueLyser II (2 x 1 min)
⢠RNA was purified using the RNeasy Plant Mini Kit; analyzed on a 1.2% formaldehyde agarose gel
⢠The sharp and equally intense ribosomal RNA bands indicate reproducible purification of intact RNA
Back to basics: Fundamental concepts and special considerations in RNA isolation 31
32. Sample to Insight
Isolating RNA from bacteria and viruses
⢠Bacterial mRNA has no 5â cap and only
rarely has a poly-A tail
⢠mRNA isolation by hybrid capture is
impossible
⢠The RNeasy Protect Bacteria Kit makes
bacterial gene expression studies possible
⢠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
Amplification of RNA from plasma
⢠RT-PCR products of a 1026 nt RNA fragment
purified from plasma
⢠Serial tenfold dilutions were added to plasma
and purified using the QIAamp Viral RNA Mini Kit
Back to basics: Fundamental concepts and special considerations in RNA isolation 32
Gene
expressed
(%)
33. Sample to Insight
Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
Step 1: Sample disruption and homogenization4.1
Step 2: Isolating RNA from different samples â tips and tricks4.2
Step 3: gDNA removal for sensitive applications4.3
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 33
34. Sample to Insight
Removal of genomic DNA contamination
⢠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
⢠Eliminate genomic DNA contamination either during RNA purification or just prior
to cDNA synthesis
⢠Primer design
⢠DNase digestion (with or without DNase Booster)
⢠gDNA Eliminator Column â RNeasy Plus Kits
⢠gDNA Eliminator Solution â RNeasy Plus Universal Kit
⢠The gDNA Eliminator Solution is a novel non-enzymatic solution, which reduces
gDNA contamination of the aqueous phase. It does not contain DNase
Why is gDNA removal important?
QIAGEN solutions:
Back to basics: Fundamental concepts and special considerations in RNA isolation 34
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Effective elimination of genomic DNA contamination
The âRT curves demonstrate that RNA purified using the RNeasy Plus Mini Kit was
virtually free of genomic DNA
⢠Total RNA was purified from Jurkat cell samples
⢠1 x 106 cells per sample
⢠A: RNeasy Plus Mini Kit
⢠B: An RNA purification kit with integrated genomic DNA removal from Supplier AV
⢠Duplicate real-time RT-PCR assays for β-actin were performed with (+RT) or without (-RT)
reverse transcriptase
Back to basics: Fundamental concepts and special considerations in RNA isolation 35
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QIAGEN RNA Purification Kits
Back to basics: Fundamental concepts and special considerations in RNA isolation
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Upgrade your expectations â RNeasyPlus Kits
RNeasy Plus Procedure â with the gDNA Eliminator spin column
Back to basics: Fundamental concepts and special considerations in RNA isolation 37
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Upgrade your expectations â RNeasy Plus Universal Kits
RNeasy Plus Universal Mini Procedure - with the gDNA Eliminator Solution
Back to basics: Fundamental concepts and special considerations in RNA isolation 38
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Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 39
40. Sample to Insight
miRNA isolation technologies
miRNeasy Kits â efficient purification of miRNA from different starting materials
⢠Cells and tissues â miRNeasy Mini Kit
⢠FNB â miRNeasy Micro Kit
⢠FFPE â miRNeasy FFPE Kit
⢠Blood â miRNeasy Serum/Plasma Kit
⢠Serum/plasma â miRNeasy Serum/Plasma Kit
⢠Highly pure RNA without phenol carryover
Back to basics: Fundamental concepts and special considerations in RNA isolation 40
OD
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Exosome isolation technologies
Lower the risk of missing your biomarker!
exoRNeasy
⢠Exosomal (EVs) RNA isolation
⢠Serum, plasma, CSF and cell culture
⢠Saliva â tested by customer; no
protocol developed
exoEasy
⢠Exosome (EVs) isolation
⢠Serum, plasma and cell culture
Back to basics: Fundamental concepts and special considerations in RNA isolation 41
42. Sample to Insight
Agenda
RNA universe â RNA types and their role in cells1
Handling RNA â General remarks2
Stabilization of RNA in sample3
RNA isolation â Special considerations4
miRNA and exosome RNA purifications5
QIAGEN solutions for quality RNA6
Back to basics: Fundamental concepts and special considerations in RNA isolation 42
43. Sample to Insight
RNA Selection Wheel
Back to basics: Fundamental concepts and special considerations in RNA isolation
RNA Selection Wheel
44. Sample to Insight
RNA resource center
www.qiagen.com/qdm/rna/resources/
Back to basics: Fundamental concepts and special considerations in RNA isolation
45. Sample to Insight
Thank you for your attention!
Questions? qiawebinars@qiagen.com
Contact QIAGEN: www.qiagen.com/about-us/contact/global-contacts/
Back to basics: Fundamental concepts and special considerations in RNA isolation 45