Anda di halaman 1dari 172

RNAi AND EPIGENETICS

SOURCEBOOK

RNAi AND EPIGENETICS SOURCEBOOK

% RNA remaining

Quick start guide

1. Decide o n e ffector m olecule: s iRNA o r v ector— Chapter 1

2. Find y our g ene o nline: www.invitrogen.com/findyourgene (shown a t r ight)

3. Choose d elivery v ehicle: t ransfection r eagent, m echanical method, o r v iral d elivery— Chapter 3

4. Decide o n c ontrols— Chapter 2

5. Validate to m easure l oss o f f unction— Chapter 11

2 5. Validate to m easure l oss o f f unction— Chapter 11 Steps of

Steps of an siRNA experiment

Have o n h and:

Transfection/electroporation a gent a nd p rotocol

Assays to a ssess k nockdown a nd o ther R NAi e ffect(s)

Positive a nd n egative c ontrol s iRNAs

Two o r m ore s iRNAs to g ene o f i nterest

1
1

Find and order siRNAs at:

www.invitrogen.com/RNAi

t 2 Plate cells and transfect siRNAs 3 Prep RNA 3' 5' 5' 3' 4
t
2
Plate cells and
transfect siRNAs
3 Prep RNA
3'
5'
5'
3'
4
Monitor siRNA-induced knockdown to:
4

Validate the siRNA

Monitor transfection efficiency

Observe/measure phenotypic change

100 10 80 1 60 40 0.1 30 20 0.01 0 NEK4-1 NEK4-2 NEK4-3 TGFBR2-1
100
10
80
1
60
40
0.1
30
20
0.01
0
NEK4-1
NEK4-2
NEK4-3
TGFBR2-1
TGFBR2-2
TGFBR2-3
STK6-1
STK6-2
STK6-3
RPS6KA4-1
RPS6KA4-2
RPS6KA4-3
MAP2K1-1
MAP2K1-2
MAP2K1-3
MAP2K4-1
MAP2K4-2
MAP2K4-3
WEE1-1
WEE1-2
WEE1-3
PK428-1
PK428-2
PK428-3
STK12-1
STK12-2
STK12-3
PCNA gold
Rn
Control siRNA Survivin siRNA
Control siRNA
Survivin siRNA

0

5

10

15

20

25

30

35

40

 

Cycle

Figure 1. RNAi workflow diagram. RNAi e xperiment w orkflow f ollowing s iRNA d esign a nd s ynthesis.

Glossary of common RNAi terms

RNAi

Ribonucleic a cid i nterference (first u sed b y A . F ire a nd C . M ello e t a l., 1998).

siRNA

Short i nterfering R NA. s iRNAs a re 21–25 b p d sRNA w ith d inucleotide 3 ’ o verhangs t hat a re p rocessed f rom l onger d sRNA b y D icer i n t he R NA i nter- ference p athway. I ntroduction o f s ynthetic s iRNAs c an i nduce R NA i nterference i n m ammalian c ells. s iRNAs c an a lso o riginate f rom e ndogenous precursors.

shRNA

Short h airpin R NA; a lso s hort i nterfering h airpin. s hRNAs a re u sed i n v ector-based a pproaches f or s upplying s iRNA to c ells to p roduce s table gene s ilencing. A s trong Pol I II t ype p romoter i s u sed to d rive t ranscription o f t arget a s equence d esigned to f orm h airpins a nd l oops o f v ariable length, w hich a re p rocessed b y c ellular s iRNA m achinery. O nce i n t he c ell t he s hRNA c an d ecrease t he e xpression o f a g ene w ith c omplementary sequences b y R NAi.

miR RNAi

Vectors t hat e xpress m icroRNAs f or R NAi. m iRNAs a re 19–23 n t s ingle-stranded R NAs, o riginating f rom s ingle-stranded p recursor t ranscripts t hat are c haracterized b y i mperfectly b ase-paired h airpins. m iRNAs f unction i n a s ilencing c omplex t hat i s s imilar, i f n ot i dentical, to R ISC.

Chemically modified siRNA

siRNA m olecules w hich h ave c hemical m odifications.

RISC

RNA-induced s ilencing c omplex ( RISC). A n uclease c omplex, c omposed o f p roteins a nd s iRNA, t hat t argets a nd c leaves e ndogenous m RNAs complementary to t he s iRNA w ithin t he R ISC c omplex.

Off-target effects

Effects t hat o ccur w hen o ne o r a f ew n ontarget g enes n ot s pecifically t argeted s how l oss o f g ene f unction f ollowing t he i ntroduction o f a n s iRNA or d -siRNA p ool. T he e ffect m ay b e m ediated b y t he s ense s trand o f a n s iRNA, w hich m ay i nitiate a l oss-of-function r esponse f rom a n u nrelated gene. O ff-target e ffects c an a lso o ccur a s a s econdary e ffect o f t he a ntisense s trand o f a s pecific s iRNA i f i t h as s ufficient h omology to k nock d own the e xpression o f a n ontarget g ene.

X

Contents

Contents

SECTION I—RNA INTERFERENCE

CHAPTER 1

Contents

Introduction to RNAi

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. 2

Make y our R NA i nterference e xperiments s imple, s tress-free, a nd s uccessful

CHAPTER 2

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2

A b rief h istory o f R NAi How R NAi w orks Eight t ips f or a s uccessful s iRNA e xperiment Considerations Interview w ith G regory H annon

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. 4

Controls for RNAi experiments

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. 7

Transfection c ontrols Negative c ontrols Positive c ontrols Downstream c ontrols Interferon c ontrols Use m ultiple s iRNA s equences p er t arget to v erify r esults .

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CHAPTER 3

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. 7

. 8

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. 9

. 9

. 9

Titrate s iRNA Rescue e xperiments

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. 9

Delivering RNAi to cells—transfection and viral delivery

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. 10

Methods to a chieve h igh t ransfection e fficiency

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. 10

Importance o f m inimizing t ransfection-mediated c ytotoxicity

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. 10

Significance o f r educing o ff-target e ffects Cell h ealth Culture c onditions Passage n umber siRNA q uality siRNA q uantity Choice o f t ransfection a gent Volume o f t ransfection a gent Exposure to t ransfection a gent/siRNA c omplexes Presence o f s erum i n t he m edium d uring t ransfection Optimizing t ransfection e xperiments

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www.invitrogen.com

. . . . . . . 11 . 14 . 14 . 14 . 15

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. . . . . . . 11 . 14 . 14 . 14 . 15

SectionRNAi andIII Epigenetics Sourcebook

CHAPTER 4

Vector-based RNAi technologies

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. 17

Introduction to a denoviral a nd l entiviral R NAi v ectors f or d elivery Lentiviral a nd a denoviral R NAi v ectors—choose a ny c ell t ype f or R NAi

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. 17

 

18

CHAPTER 5

In vivo RNAi

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. 19

RNAi m olecules f or in vivo applications Choosing R NAi e ffector m olecules Choosing R NAi p urity Tracking d elivered d uplexes In vivo R NAi p rotocols Measuring R NA c oncentration Harvesting t issue—RNA e xtraction f rom t issue . Sectioning t issue Protein e xtraction f rom t issues Frequently a sked q uestions a bout in vivo R NAi .

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. 19

. 19

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