Nielsen H. RNA: Methods and Protocols

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204 Henriksen et al.

12. There is not one single housekeeping gene suitable for all

experiments. It is recommended to test the consistency

of expression of several housekeeping genes in your own

experiments. We have used the Human Endogenous Con-

trol Gene Panel (tataa Biocenter) to ﬁnd the housekeep-

ing genes most suitable for our experimental setup. We

always use 2–4 different housekeeping genes in each real-

time PCR experiment.

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Chapter 14

Validation of RNAi by Real Time PCR

Knud Josefsen and Ying C. Lee

Abstract

Real time PCR is the analytic tool of choice for quantiﬁcation of gene expression, while RNAi is

concerned with downregulation of gene expression. Together, they constitute a powerful approach in

any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify

luciferase mRNA silencing.

Key words: RNA isolation, reverse transcription, real time PCR, LightCycler, luciferase, shRNA,

RNAi.

1. Introduction

Real time PCR and RNAi (1) together constitute a powerful

approach in any loss of function studies of selective genes. Real

time PCR is essentially a cycle by cycle kinetic analysis of a signal

generated by a ﬂuorescent reporter used in the ampliﬁcation reac-

tion. It detects the DNA ampliﬁcation during the PCR reaction

with a b road dynamic range, and analyses each sample at its opti-

mal point of ampliﬁcation. The increase in ﬂuorescence is directly

proportional with the amount of PCR product accumulated in

the reaction. The threshold cycle (C

) is the key parameter in real

time PCR and is deﬁned as the cycle number when measurable

ampliﬁcation product has accumulated. Thus, the more speciﬁc

cDNA a sample contains, the faster the ampliﬁcation reaches the

detection level and the lower C

is obtained.

The most cost-effective and simplest detection chemistry for

real time PCR is SYBR Green I dye. SYBR Green I ﬂuoresce when

bound to double-stranded DNA (dsDNA), whereas it only binds

H. Nielsen (ed.), RNA, Methods in Molecular Biology 703,

DOI 10.1007/978-1-59745-248-9_14, © Springer Science+Business Media, LLC 2011

205

206 Josefsen and Lee

single-stranded DNA poorly. Unlike other detection chemistries

used in real time PCR, SYBR Gr een I can present a speciﬁcity

problem because the dye binds to any dsDNA formed during the

real time PCR run. However, melting curve analysis can be used

to discriminate the speciﬁc product from non-speciﬁc products

such as primer-dimers, which may form in the reaction. Melt-

ing curve analysis determines the T

of the PCR product, which

is deﬁned as the temperature where 50% of the DNA is single-

stranded. T

can form the basis for the inclusion of a 4th segment

in the PCR ampliﬁcation program if non-speciﬁc ampliﬁcation is

present, since it reduces the non-speciﬁc ﬂuorescence signal gen-

erated in real time PCR (2). The 4th segment temperature is usu-

allysetat3–5

◦

C lower than the T

for the speciﬁc product.

An important consideration for a successful real time PCR

result is the precision of real time PCR cycler. Although replicates

may improve the reliability of the results, in particular when quan-

tifying targets with low copy number, surprisingly little attention

is usually devoted to the fact that different real time PCR cyclers

operate with very variable precision from virtually no variability

to more than 100% variation (see Note 1). Equally important is

the primer design. Software is available on the web which will

optimise factors like length and G/C content of the primers as

well as prevent secondary structures and overlapping sequences.

Likewise, for reliable and accurate real time data it is impera-

tive to use high-quality RNA (3), optimised PCR master mix

and reagent cocktails as well as paying attention to mixing all

reagent components thoroughly during the set-up of the exper-

iments. Finally, any comparison of gene expression between dif-

ferent samples invariably also involves quantiﬁcation of a reference

gene in all samples (4). These general requirements for successful

real time PCR procedures and possible pitfalls have been detailed

in a recent article by Nolan et al. (5). Further information can be

found on the authors’ website (www.realtimepcr.dk).

All of the above-mentioned factors can inﬂuence real time

PCR efﬁciency which is deﬁned as the proportion of target ampli-

con, which is ampliﬁed from one cycle to the next. To determine

the efﬁciency (E) of the reaction, C

is plotted against the log-

arithm of the dilution of the template (6). In this graph, 100%

efﬁciency corresponds to a slope of –3.3, and 90% or 110% to

–3.6 or –3.1, respectively, since E = 10

–1/slope

. Calculation of

the real time PCR efﬁciency is of particular importance when the

delta/delta method is employed to compare samples; such data

is only valid when the ampliﬁcation efﬁ ciencies of the target and

reference genes are equal (5).

Single-stranded antisense RNAs have been demonstrated to

inhibit effectively gene expression in mammalian systems (7).

The production of single-stranded RNA is initiated by the action

of dicer, a RNase III class enzyme acting upon endogenous

Validation of RNAi by Real Time PCR 207

double-stranded RNAs (dsRNAs) or exogenously introduced

short dsRNAs, and ﬁnalised in the RNA-induced silencing com-

plex (RISC), within which the RNA sense strand is degraded,

whereas the antisense RNA strand can act as a guide sequence to

mediate speciﬁc cleavage of targeted mRNA, resulting in RNAi.

The length of duplex siRNA molecules is usually limited to less

than 30 nucleotides (8) as non-speciﬁc effects such as cellular

interferon activation may otherwise occur. In addition, vector-

encoded RNAi, shRNAs (short-hairpin RNAs), have also been

developed for long-term gene silencing experiments.

The success of any RNAi experiment depends on effective

design of siRNAs or shRNAs, ef ﬁcient delivery to the cells, an

appropriate phenotypic assay to assess the RNAi effect and equally

important: an assay for the target gene silencing. As RNAi exerts

its effects at the mRNA level, the simplest, quickest and most

sensitive assay for RNAi validation is real time PCR. Here, we

demonstrate the use of real time PCR to validate the silencing of

luciferase gene expression by shRNA. Hela cells were transfected

with either luciferase shRNA construct or with a negative control

shRNA. This was followed by isolation of total RNA, ﬁrst strand

cDNA synthesis and real time PCR by LightCycler. The result

showed that luciferase mRNA was reduced fourfold in cells trans-

fected with the luciferase shRNA construct compared with cells

transfected with negative control shRNA.

2. Materials

2.1. Tissue Culture

1. Hela cells (American Tissue Culture Collection).

2. RPMI 1640 and Trypsin/EDTA (Lonza).

3. Foetal bovine serum, OptiMEM and Lipofectamine 2000

(Invitrogen).

4. 90% RMI1640 + 10% foetal bovine serum

5. T75 tissue culture ﬂasks and 12-well tissue cultur e plates

(Costar).

6. Plasmids pShag+ which encodes shRNA against luciferase

and pShag-, a negative control, were supplied by Chris-

ter Einvik, Tromsø, Norway (9). pGL3-control encoding

luciferase and pGL3 basic, a negative control (Promega).

2.2. RNA Isolation

(see Note 2)

1. Trizol (Invitr ogen).

2. Chloroform, isopropanol and ethanol (Merck Chemicals).

3. TE buffer: 1 mM Tris-HCl, pH 7.0, 10 mM EDTA.

4. UV spectrophotometer (BioPhotometer: Eppendorf).

208 Josefsen and Lee

2.3. Reverse

Transcription

1. Reverse transcriptase kit (iScript from Bio-Rad) (see Note 3).

2.4. Real Time PCR

1. Real time PCR mix (FastStart

plus

DNA Master SYBR Green

I; Roche) (see Note 4).

2. PCR Primers (Sigma Genosys, England); puriﬁcation: DST

(see Note 5). Luciferase (fragment sized 201 bp), forward:

ATG AAC GTG AAT TGC TCA ACA G, reverse: TAA AAC

CGG GAG GTA GAT GAG A. Reference gene: Ribosomal

protein L13 (RPL13, 227 bp product), forward: CCA CCC

TAT GAC AAG AAA AAG C, reverse: ACA TTC TTT TCT

GCC TGT TTC C.

3. Real time PCR cycler (LightCycler version 1.5 with Light-

Cycler Software Version 3.5.3; Roche Molecular Biochemi-

cals).

4. Reaction capillaries (Roche) (see Note 6).

5. TBE (Qiagen) and agarose (Invitrogen).

6. TOPO TA cloning kit and One Shot Competent E.

coli (Invitrogen) for subcloning of PCR fragments (see

Note 7).

3. Methods

3.1. Tissue Culture

1. Grow Hela cells in 90% RMI1640 + 10% foetal bovine serum

in T75 bottles with medium replacements every other day

(see Note 8).

2. Split cells when they reach approximately 70% conﬂu-

ence: remove the medium, rinse the cells brieﬂy in 2 mL

trypsin/EDTA, remove it and add 2 mL of fresh

trypsin/EDTA. Leave it on the cells until they detach when

the bottle is tilted. Add 8 mL of medium and triturate the

cells (pipette the medium in and out of a 13-mL pipette

8–10 times until most of the cells are suspended as single

cells). Transfer to 2–3 new T75 bottles and add medium to

approximately 13 mL per bottle.

3. For transfection, split cells as above, but transfer the cells to

6-well plates to obtain a conﬂuence of approximately 60%

after 24 h of incubation.

4. For each transfection, mix 1.8 μg plasmid with 250 μL

serum-free DME in a sterile Eppendorf tube and 3 μLLipo-

fectamine 2000 and 250 μL OptiMEM in another. Com-

bine the content of the two tubes and leave for 20 min

to form complexes. You need three transfections: pGL3

Validation of RNAi by Real Time PCR 209

basic + pShag(–), pGL3 control + pShag- and pGL3 con-

trol + pShag+.

5. Replace medium on the cells (1.5 mL) and add the transfec-

tion mixtures to the cells.

6. Harvest the cells 48 h later.

3.2. RNA Isolation

1. Remove medium, and add 1 ml of cold (on ice) Trizol per

well. Shake gently for 5 min at room temperature.

2. Transfer lysate to Eppendorf tubes and add 200 μL of chlo-

roform.

3. Shake vigorously, and leave on ice for 20 min. Shake occa-

sionally during this time.

4. Centrifuge for 30 min at 15,000×g.

5. Recover top (aqueous) phase.

6. Add 0.5 mL isopropanol. Leave at –20

◦

C for 30 min.

7. Centrifuge for 30 min at 15,000×g and remove super-

natant. Watch the pellet (see Note 9).Add1mL75%

ethanol and remove it again. Spin the tube for a few sec-

onds and r emove the remaining drops of ethanol.

8. Air dry for 5–10 min with open lid.

9. Redissolve the RNA in 20 μL RNAse-free water or 5 mM

EDTA.

10. To quantify the RNA, dilute 1 μLofRNAin99μLof

TE and measure the absorption at 260 and 280 nm. The

260/280 should be around 1.8–2 for high-quality RNA,

lower with increasing amounts of protein contamination

(aromatic amino acids absorbs at 260 nm). Calculate your

stock RNA concentration in μg/μLasOD

260

× 40 × 0.1

m/1 μL (light path = 0.1 cm).

3.3. First Strand

cDNA Synthesis

1. Prepare master-mix for multiples samples, and add RNA last.

Reaction can be scaled up when large amounts of input RNA

is used (30 μL for 1.5 μg).

Component Volume per reaction

5X iScript buffer 3 μL

iScript reverse transcriptase 0.75 μL

Nuclease-free water 1.25 μL

RNA template (up to 0.75 μg

total RNA) (see Note 10)

10 μL

15 μL

210 Josefsen and Lee

2. Incubate reaction mix in a thermocycler:

◦

C5min

◦

C30min

◦

C5min

◦

C hold

3.4. Real Time PCR

3.4.1. Setting Up the

Reaction

1. Cool rotor and centrifuge bucket at –20

◦

2. Prepare stock of master reaction mix on ice. Protect from

light until needed. For each reaction you will need

Water 5 μL

Primers ( see Note 11) (stock with each

10 μM)

1 μL

cDNA 2 μL

5× FastStart

plus

DNA Master SYBR

Green I (Roche)

2 μL

Prepare one sample per cDNA to be analysed (see

Note 12), eight samples for the standard curve (if you run

consecutive runs, you only need one standard in each of the

following rotors), and two negative controls: one (cDNA-)

negative contr ol to which no cDNA is added and one

(RNA-) negative contr ol where no RNA was added to the

reverse transcription reaction.

3. Insert rotor into centrifuge bucket and put the bucket

on ice.

4. Insert the capillaries into the rotor.

5. Pipette 8 μL of the re action mix into each capillary (see

Note 13).

6. Pipette 2 μL of cDNA into each capillary.

7. Cap the capillaries.

8. Spin the rotor in the special centrifuge which comes with

the LightCycler.

9. Start execution of the program using the following

parameters:

slope (

◦

C/s)

Validation of RNAi by Real Time PCR 211

Pre -incubation

Segment 1 Denaturation 95

◦

C, 600 s 20

Ampliﬁcation (40 cycles)

Segment 1 Denaturation 95

◦

C, 10 s 20

Segment 2 Anneal 60

◦

C,5s 20

Segment 3 Extension 72

◦

C, 15 s 20

Acquisition (Single mode)

Melting curve

Segment 1 95

◦

C0s 20

Segment 2 65

◦

C15s20

Segment 3 95

◦

C0s 0.1

Cooling

Segment 1 40

◦

C30s20

10. Repeat 1–9 using the housekeeping primer pair (see

Note 14).

3.4.2. Preparation of the

Real Time PCR Standard

1. PCR amplify a 150- to 250-bp fragment of your control

gene using your preferred method.

2. Verify that the fragment has the corr ect size by agarose gel

electrophoresis.

3. Subclone fragment:

PCR product (fresh ideally) 3 μL

Water 1 μL

Salt solution 1 μL

TOPO vector 1 μL

Total volume 6 μL

Mix gently in tube using the pipette tip; incubate up to

30 min at room temperature (5 min r ecommended by

manufacturer).

4. Place reaction tube on ice.

5. Thaw One Shot Competent cells (stored at –80

◦

C) on ice.

6. Add 2 μL of the TOPO reaction to 40 μL of bacteria, and

mix gently using pipette tip or ﬂicking.

7. Incubate on ice for 15–30 min.

8. Heat-shock the cells for 45 s at 42

◦

C, and place tube(s) on

ice immediately.

212 Josefsen and Lee

9. To each vial (tube), add 250 μL room temperature SOC

medium and cap and shake the tube at 37

◦

C for 1 h (using

Eppendorf Ther mal Mixer).

10. Spread 10–50 μL from each transformation onto LB agar

plate (imMedia AMP Blue from Fluka).

11. Incubate overnight at 37

◦

12. Pick white (or light blue) colonies, culture them over

night in LB medium using Eppendorf LidBac, with Eppen-

dorf Thermal Mixer at 37

◦

C, 1,200 rpm and isolate plas-

mid DNA by a method which pr oduces DNA suitable

for sequencing (usually by afﬁnity chromatography as for

instance the Wizard kit from Promega or the FastPlasmid

Mini from Eppendorf).

13. Linearise the plasmids:

Plasmid 2 μL

Restriction buffer 2 μL

EcoRI 1 μL

Water 5 μL

Incubate at 37

◦

Cfor1h.

14. Analyse by 3% agarose gel electrophoresis at 50 V for

20 min (minigel). If the fragments are of the expected

size, sequence the plasmid to verify that the expected DNA

sequence has been introduced into the p lasmid.

15. Upon veriﬁcation of sequence, prepare a larger amount of

linearised plasmid (as above, using 10 – 10 – 2 – 78 μL,

respectively).

16. Check for completion of the reaction by analysing 5 μLof

the reaction mixture by 3% agarose gel electrophoresis. If

the reaction is not complete, extend the incubation and/or

add additional restriction enzyme.

17. Inactivate the enzyme by incubating for 10 min at 65

◦

18. Store the digestion at –20

◦

C for future use.

19. Prepare dilution standard:

• Label eight tubes 1–8. Add 999 μL of water to tube 1

and 900 μL of water to tubes 2–8.

• Transfer 1 μL of digestion reaction to tube 1. Mix thor-

oughly using a vortex mixer.

• Transfer 100 μL to successive tubes, each time ensuring

thorough mixing before transfer.

Validation of RNAi by Real Time PCR 213

• Use the dilutions as a real time PCR standard: Substitute

the 2 μL of cDNA normally used in the real time PCR

reaction for 2 μL of the content of tubes 1–8.

• You may use the dilutions for the day, but otherwise

make up fresh dilutions each time you want to use the

standard.

3.4.3. Analysing

the Results

1. For any new ampliﬁcation, analyse a representative number

of reactions by 3% agarose TBE gel electrophoresis. Verify

that the resulting band is of the expected size and that other

products are not present.

2. Check the melting curve analysis. For a 150–250 bp

gene, you would expect a melting temperature of approx-

imately 80–85

◦

C. Curves showing melting temperatures

around 60–65

◦

C are almost certainly unspeciﬁc products

like primer-dimer ampliﬁcations.

3. Check your (cDNA−) negative control. There should be

no ampliﬁcation, or the C

should be much higher than

reactions containing cDNA. If you obtain a C

from the

(cDNA−) negative control, consider if the ampliﬁcation

stems from non-speciﬁc ampliﬁcation, and try to r epeat the

reaction with an additional step (4th segment) after the

extension step. The temperature should be above the melt-

ing temperature of primer-dimers and below the melting

temperature of your speciﬁc product.

4. Check your (RNA−) negative control. There should be no

ampliﬁcation. If there is, your chemicals might be contam-

inated, or your RNA might contain genomic DNA and

would need DNAse treatment before being used in the real

time PCR reaction.

5. Calculate the expression of your speciﬁc mRNA relative to

the expression of your housekeeping gene to normalise for

variation in the amount of cDNA transferred to the real time

PCR reaction or variable efﬁciencies in reverse transcription.

3.4.4. Example

of Results

Using the above method, cDNA samples from Hela cells trans-

fected with pGL3 control plasmid encoding luciferase and either

pSHAG− (control) or pSHAG+ (coding for shRNA against the

luciferase gene) were analysed by the absolute quantiﬁcation

(see Fig. 14.1). Representative results are shown: A: standard

curve. B: samples in duplicate. The three sets of curves represent

(left to right): cells transfected with pGL3 control, pGL3 con-

trol/pSHAG+ and pGL3 basic, respectively. C: relative luciferase

gene expression normalised against RPL13 which was used as a

housekeeping gene. The shRNA induced a fourfold knockdown

of luciferase gene expression.