Nielsen H. RNA: Methods and Protocols

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184 Sioud

antagonize with immunostimulatory RNA to activate

indoleamine 2,3-dioxygenase, an immunosuppressive pro-

tein, in human monocytes (48). In accordance with our

data, Robbins and colleagues have r eported that 2



-modiﬁed

immunostimulatory RNAs can function as TLR7/8 antagonist

by inhibiting TLR7 signaling by immunostimulatory RNAs and

loxoribine in both murine and human cells (49). Suppressive



-modiﬁed RNAs should represent a new class of agents that

may be useful in the treatment of autoimmunity triggered by

TLR7/8 signaling. Some studies have described a number of

different classes of non-stimulatory DNA sequences including

mutated CpG sequences and repeats of the TTAGGG motifs

present in mammalian telomeres (50). Interestingly, the linkage

of a G-rich DNA oligonucleotide (10-mer) to a 2



-modiﬁed

immunomodulatory RNA (10-mer) led to the design of a

sequence capable of blocking TLR7, TLR8, and TLR9 signaling

(Sioud M, unpublished data). The development of single tri-

functional oligonucleotide may be required for the treatment of

autoimmune disease where TLRs are involved.

8. Sequence-

Dependent

Off-Target Effects

When ﬁrst reported, siRNAs seemed highly speciﬁc, as a single

mutation in the target site could be demonstrated to completely

abolish silencing (5). However, it was soon demonstrated that

mutations in the target site of several siRNAs did not always

inhibit knockdown (18). Furthermore, siRNAs with only par-

tial complementarily to mRNAs can also cause a reduction in the

RNA levels of a large number of transcripts (15). Another poten-

tial sour ce of siRNA toxicity is therefore the destruction of cellu-

lar mRNAs that share partial homology to the siRNA sequences.

Because the cellular pathways activated by miRNAs and siRNAs

are overlapping, it is more likely that each siRNA sequence will

exhibit a miRNA activity. The most commonly used strategy

to ensure siRNA target speciﬁcity is the basic local alignment

search tool BLAST. However, short sequence stretches may not

be detected by BLAST program. In addition, the identiﬁcation

of such sequences does not necessarily indicate the occurrence of

off-target effects. Similarly, the absence of short homologies will

not rule out off-target effects. The best way to deal with this prob-

lem is to analyze global gene expression, speciﬁcally when siRNAs

are going to be used in functional genomics or to develop ther-

apeutics. During our studies with siRNAs, we have found that



-uridine modiﬁcations of siRNAs not only evade the activa-

tion of TLR7/8 but they can block most of TLR-independent

Promises and Challenges in Developing RNAi as a Research Tool and Therapy 185

effects including off-target effects (36). Although the evading

mechanisms remain to be investigated, it is probable that the

interaction of siRNA sequences with unintended cellular mRNAs

is affected by chemical modiﬁcations. Modiﬁcations of RNA

might be particularly disruptive for siRNA binding to mismatched

sequences. In accordance with these observations, Jackson and

colleagues found that the incorporation of 2



-O-methyl group at

the second position of the siRNA guide strand can reduce most

off-target gene silencing effects without affecting siRNA silenc-

ing of the intended target gene (51). Collectively, these studies

offer a simple strategy for reducing off-target effects. Therefore,

I suggest the incorporation of few 2



-modiﬁed bases in siRNA

sequences, particulary thymidines.

References

1. Fire, A., Xu, S., Montgomery, M. K., Kostas,

S. A., Driver, S.E, Mello, C. C. (1998)

Potent and speciﬁc genetic interference by

double-stranded RNA in Caenorhabditis ele-

gans. Nature 391, 806–811.

2. Jorgensen, R. (1990) Altered gene expres-

sion in plants due to trans interactions

between homologous genes. Trends Biotech-

nol 8, 340–344.

3. Hamilton, A.J, Baucombe, D. C. (1999) A

species of small antisense RNA in posttran-

scriptional gene silencing in plants. Science

286, 950–952.

4. Sen, G. C. (2001) Viruses and interferons.

Annu Rev Microbiol 55, 255–281.

5. Elbashir, S. M., Harborth, J., Lendeckel,

W., Yalcin, A., Weber, K., Tuschl, T. (2001)

Duplexes of 21-nucleotide RNAs mediate

RNA interference in cultured mammalian

cells. Nature 411, 494–498.

6. Sioud, M. (2004) Therapeutic siRNAs.

Trends Pharmacol Sci 25, 22–28.

7. Hannon, G. J., Rossi, J. J. (2004) Unlock-

ing the potential of the human genome with

RNA interference. Nature 431, 371–378.

8. Liu, J., Carmell, M. A., Rivas, F. V., Marsden,

C. G., Thomson, J. M., Song, J. J., Ham-

mond, S. M., Joshua-Tor, L., Hannon, G. J.

(2004) Argonaute2 is the catalytic engine of

mammalian RNAi. Science 305, 1437–1441.

9. Song, J. J., Smith, S. K., Hannon, G. J.,

Joshua-Tor, L. (2004) Crystal str ucture of

Argonaute and its implications for RISC

slicer activity. Science 305, 1434–1437.

10. Matranga, C., Tomari, Y., Shin, C., Bartel,

D. P., Zamore, P. D. (2005) Passenger-strand

cleavage facilitates assembly of siRNA into

Ago2-containing RNAi enzyme complexes.

Cell 123, 607–620.

11. Rand, T. A., Petersen, S., Du, F., Wang,

X. (2005) Argonaute2 cleaves the anti-guide

strand of siRNA during RISC activation. Cell

123, 621–629.

12. Ma, J. B., Yuan, Y. R., Meister, G., Pei, Y.,

Tuschl, T., Partel, D. J. (2005) Structural

basis for 5



-end-speciﬁc recognition of guide

RNA by the A. fulgidus Piwi protein. Nature

434, 666–670.

13. Lee, Y. S., et al. (2004) Distinct roles

for Drosophila Dicer-1 and Dicer-2 in the

siRNA/miRNA silencing pathway. Cell 117,

69–81.

14. Sioud, M., Sørensen, D. R. (2003) Cationic

liposome-mediated delivery of siRNAs in

adult mice. Biochem Biophys Res Commun

312, 1220–1225.

15. Jackson, A. L., Bartz, S. R., Schelter, J.,

Kobayashi, S. V., Burchard, J., Mao, M., Li,

B., Cavet, G., Linsley, P. S. (2003) Expres-

sion proﬁling reveals off-target gene regula-

tion by RNAi. Nat Biotechnol 21, 635–637.

16. Semizarov, D., Frost, L., Sarthy, A., Kroeger,

P., Halbert, D. N., Fesik, S. W. (2003)

Speciﬁcity of short interfering RNA deter-

mined through gene expression signatures.

Proc Natl Acad Sci USA 100, 6347–6352.

17. Sledz, C. A., Holko, M., de Veer, M. J.,

Silverman, R. H., Williams, B. R. (2003)

Activation of the interferon system by short-

interfering RNAs. Nat Cell Biol 5, 834–839.

18. Holen, T., Amrzguioui, M., Wiiger, M. T.,

Babaie, E., Prydz, H. (2002) Positional

effects of short interfering RNAs targeting

the human coagulation tissue factor. Nucleic

Acids Res 30, 1757–1766.

19. Schwarz, D. S, et al. (2003) Asymmetry in

the assembly of the RNAi enzyme complex.

Cell 115, 199–208.

186 Sioud

20. Khvorova, A., Reynolds, A., Jayasena, S.

D. (2003) Functional siRNAs and miRNAs

exhibit strand bias. Cell 115, 209–216.

21. Ui-Tei, K., Naito, Y., Saigo, K. (2007)

Guidelines for the selection of effectives

short-interferin RNA sequences for func-

tional genomics. Methods Mol Biol 361,

201–216.

22. Li, S., Peters, G. A., Ding, K., Zhang, X.,

Qin. J., Sen, G. C. (2006) Molecular basis

for PKR activation by PACT or dsRNA. Proc

Natl Acad Sci USA 103, 1005–1010.

23. Kato, H., Takeuchi, O., Sato, S., Yoneyama,

M., Yamamoto, M., Matsui, K., Uematsu, S.,

Jung, A., Kawai, T., Ishii, K. J., Yamaguchi,

O., Otsu, K., Tsujimura, T., Koh, C. S., Reis

e Sousa, C., Matsuura, Y., Fujita, T., Akira, S.

(2006) Differential roles of MDA5 and RIG-

I helicases in the recognition of RNA viruses.

Nature 441, 101–105.

24. Meylan, E., Tschopp, T., Karin, M. (2006)

Intracellular pattern recognition receptors in

the host response. Nature 442, 39–44.

25. Takeda, K., Akira, S. (2005) Toll-like recep-

tors in innate immunity. Int Immunol 17,

1–14.

26. Sioud, M. (2005) Innate sensing of self and

non-self RNAs by Toll-like receptors. Trends

Mol Med 12, 167–176.

27. Alexopoulou, L., Holt, A. C., Medzhitov,

R., Flavell, R. A. (2001) Recognition of

double-stranded RNA and activation of NF-

kappaB by Toll-like receptor 3. Nature 413,

732–738.

28. Heil, F., Hemmi, H., Hochrein, H., Ampen-

berger, A., Kirschning, C., Akira, S., Lipford,

G., Wagner, H., Bauer, S. (2004) Species-

speciﬁc recognition of single-stranded RNA

via toll-like receptor 7 and 8. Science 303,

1526–1529.

29. Krieg, A. M. (2002) CpG motifs in bacterial

DNA and their immune effects. Annu Rev

Immunol 20, 709–760.

30. Cao, W., Liu, Y. J. (2007) Innate immune

functions of plasmacytoid dendritic cells.

Curr Opin Immunol 19, 24–30.

31. Kariko, K., Bhuyan, P., Capodici, J., Weiss-

man, D. (2004) Small interfering RNAs

mediate sequence-independent gene sup-

pression and induce immune activation by

signaling through toll-like receptor 3. J.

Immunol 172, 6545–6549.

32. Sioud, M. (2005) Induction of inﬂamma-

tory cytokines and interferon responses by

double-stranded and single-stranded siRNAs

is sequence-dependent and r equires endoso-

mal localization. J Mol Biol 348, 1079–1090.

33. Hornung, V., Guenthner-Biller, M.,

Bourquin, C., Ablasser, A., Schlee, M.,

Uematsu, S., Noronha, a., Manoharan, M.,

Akira, S., de Fougerolles, A., Endres, S.,

Hartmann, G. (2005) Sequence-speciﬁc

potent induction of IFN-alpha by short inter-

fering RNA in plasmacytoid dendritic cells

through TLR7. Nat Med 11, 263–270.

34. Judge, A. D., Sood, V., Shaw, J. R.,

Fang, D., McClintock, K., MacLachlan, I.

(2005) Sequence-dependent stimulation of

the mammalian innate immune response

by synthetic siRNA. Nat Biotechnol 23,

457–462.

35. Sioud, M. (2006) Single-stranded small

interfering RNA are more immunostimu-

latory than their double-stranded counter-

parts: a central role for 2



-hydroxyl uridines

in immune responses. Eur J Immunol 36,

1222–1230.

36. Cekaite, L., Furset, G., Hovig, E., Sioud,

M. (2007) Gene expression analysis in blood

cells in response to unmodiﬁed and 2



modiﬁed s iRNAs reveals TLR-dependent

and independent effects. J Mol Biol 365,

90–108.

37. Goodchild, A., Nopper, N., King, A., Doan,

T., Tanudji, M., Arndt, G. M., Poidinger, M.,

Rivory, L. P., Passioura, T. (2009) Sequence

determinants of innate immune activation by

short interfering RNAs. BMC Immunol 10,

40–48.

38. Marques, J. T., Devosse, T., Wang, D.,

Zamanian-Daryoush, M., Serbinowski, P.,

Hartmann, R., Fujita, T., Behlke, M. A.,

Williams, B. R. (2006) A structural basis

for discriminating between self and nonself

double-stranded RNAs in mammalian cells.

Nat Biotechnol 24, 559–565.

39. Bartel, D. P. (2004) MicroRNAs: genomics,

biogenesis, mechanism, and function. Cell

116, 281–294.

40. Han, J., Lee, Y., Yeom, K. H., Nam, J. W.,

Heo, I., Rhee, J. K., Sohn, S. Y., Cho, Y.,

Zhang, B. T., Kim, V. N. (2006) Molecular

basis for the recognition of primary microR-

NAs by the Drosha-DGCR8 complex. Cell

125, 887–901.

41. Furset, G., Sioud, M. (2007) Design of

bifunctional siRNAs: combining immunos-

timulation and gene-silencing in one single

siRNA molecule. Biochem Biophys Res Com-

mun 352, 642–649.

42. Hornung, V., Ellegast, J., Kim, S., Brzozka,

K., Jung, A., Kato, H., Poeck, H., Akira,

S., Conzelmann, K. K., Schlee, M., Endres.

S., Hartmann, G. (2006) 5



-Triphosphate

RNA is the ligand for RIG-I. Science 314,

994–997.

43. Morrissey, D. V., Lockridge, J. A., Shaw,

L., Blanchard, K., Jensen, K., Breen, W.,

Promises and Challenges in Developing RNAi as a Research Tool and Therapy 187

Hartsough, K., Machemer, L., Radka, S.,

Jadhav, V., Vaish, N., Zinnen, S., Vargeese,

C., Bowman, K., Shaffer, C. S., Jeffs, L.

B., Judge, A., MacLachlan, I., Polisky, B.

(2005) Potent and persistent in vivo anti-

HBV activity of chemically modiﬁed siRNAs.

Nat Biotechnol 23, 1002–1007.

44. Judge, A. D., Bola, G., Lee, A. C.,

MacLachlan, I. (2006) Design of non-

inﬂammatory synthetic siRNA mediating

potent gene silencing in vivo. Mol Ther 13,

494–505.

45. Kariko, K., Buckstein, M., Ni, H., Weiss-

man, D. (2005) Suppression of RNA recog-

nition by Toll-like receptors: the impact

of nucleoside modiﬁcation and the evo-

lutionary origin of RNA. Immunity 23,

165–175.

46. Sioud, M. (2007) RNA interference and

innate immunity. Adv Dr ug Deliv Rev 59,

153–163.

47. Sioud, M., Furset, G., Cekaite, L. (2007)

Suppression of immunostimulatory siRNA-

driven innate immune activation by



-modiﬁed RNAs. Biochem Biophys Res Com-

mun 361, 122–126.

48. Furset, G., Floisand, Y., Sioud, M. (2007)

Impaired expression of indoleamine 2, 3-

dioxygenase in monocyte-derived dendritic

cells in response to Toll-like receptor-7/8 lig-

ands. Immunology 123, 263–271.

49. Robbins, M., Judge, A., Liang, L., McClin-

tock, K., Yaworski, E., MacLachlan, I. (2007)



-O-methyl-modiﬁed RNAs act as TLR7

antagonists. Mol Ther 15, 1663–1669.

50. Zhu, F. G., Reich, C. F., Pisetsky, D.

S. (2002) Inhibition of murine dendritic

cell activation by synthetic phosphoroth-

ioate oligodeoxynucleotides. JLeukocBiol

72, 1154–1163.

51. Jackson, A. L., Burchar d, J., Leake, D.,

Reynolds, A., Schelter, J., Guo, J., John-

son, J. M., Lim, L., Karpilow, J., Nichols,

K., Marshall, W., Khvorova, A., Lins-

ley, P. S. (2006) Position-speciﬁc chemi-

cal modiﬁcation of siRNAs reduces “off-

target” transcript silencing. RNA 12,

1197–1205.

Chapter 13

Inhibition of Gene Function in Mammalian Cells Using

Short-Hairpin RNA (shRNA)

Jørn Remi Henriksen, Jochen Buechner, Cecilie Løkke,

Trond Flægstad, and Christer Einvik

Abstract

RNAi is now the preferred method for silencing gene expression in a variety of systems. In this chapter

we describe the procedure for applying short-hairpin RNA (shRNA) to study gene function. Detailed

descriptions of target site selection, shRNA construction, shRNA transfection and target knockdown

validation are included.

Key words: RNAi, short-hairpin RNA, gene silencing, target knockdown validation, MYCN,

neuroblastoma.

1. Introduction

Gene silencing by antisense technology is now being used as

a powerful molecular tool to study gene functions in living

organisms. The antisense agents bind to target messenger RNA

(mRNA), thus inactivating the target gene expression. The

inhibitory ef fects on protein production from the corresponding

gene may result in phenotypic changes. Thereby, the function of

the gene can be understood. To date, there are a number of anti-

sense molecules that can affect efﬁcient post-transcriptional gene

silencing. They include antisense oligonucleotides (ON), anti-

sense ‘third-generation’ nucleic acid analogues (Peptide Nucleic

Acid (PNA), Locked Nucleic Acid (LNA) or morpholinos),

ribozymes, small-interfering RNAs (siRNAs), short-hairpin RNAs

(shRNAs) and microRNAs (miRNAs). These antisense molecules

cause speciﬁc gene inhibitory effects through different mecha-

nisms. In this chapter, we demonstrate the characteristics of gene

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

DOI 10.1007/978-1-59745-248-9_13, © Springer Science+Business Media, LLC 2011

189

190 Henriksen et al.

silencing using RNAi-based short-hairpin RNA (shRNA) technol-

ogy.

RNAi is a highly conserved gene silencing mechanism that

plays an important role in regulation of gene expression. In addi-

tion, the RNAi system is important in pr otecting the host cell

from viral infections and invasion by mobile genetic elements (1).

The RNAi pathway takes place in the cytoplasm and can be

subdivided into two phases; an initiation and an effector phase.

In the initiation phase, double-stranded RNAs are cleaved by

the RNase III-like nuclease Dicer to produce 21–23 nt duplex

RNAs, called small interfering RNAs (siRNAs). During the effec-

tor phase, the siRNA molecule is incorporated into the RNA-

induced silencing complex (RISC), where an ATP-dependent

RNA helicase activity unwinds the duplex. The siRNA strand

which is antisense to the target RNA (guide strand) is incor-

porated into RISC, while the complementary passenger strand

is destroyed. The guide strand permits highly sequence-speciﬁc

recognition of the complementary mRNA, which is then cleaved

by Argonaute 2, a component of RISC. This results in inhibition

of protein synthesis from the mRNA (2).

Unlike miRNA and other long dsRNAs, shRNAs transcribed

by RNA pilymerase III from exogenously introduced DNA do

not require initial processing in the nucleus by the RNase III

enzyme, Drosha. These tight hairpin turn RNA structures are

transported directly to the cytoplasm via exportin-5 where they

are cleaved by Dicer into siRNA molecules, which then follow

the RNAi pathway for gene silencing (3).

RNAi is now a well-established method for high-throughput

analysis as well as for functional studies in vitro, including mam-

malian cells (4). Two different methods are commonly used to

deliver siRNA molecules for gene silencing in mammalian cell

lines: (a) synthetic siRNAs (5) and (b) RNA polymerase III tran-

scribed shRNAs from plasmids or viral vectors (6, 7).

Plasmid vector based shRNA expression is a low-cost and

easy-to-perform method for studying gene function in mam-

malian cells. In addition, this strategy offers the possibility of

inducible siRNA expression in the case where gene silencing is

expected to have deleterious effects on the target cell (8).

2. Materials

2.1. Oligonucleotides

and Plasmids Used

for Cloning

1. Oligonucleotides:

Inhibition of Gene Function in Mammalian Cells Using shRNA 191

Name Sequence (5



-3



) Description

ON 3 GTTTTCCCAGTCACGACGTTGTA M13 forward sequencing

primer

ON10 CGGGATCCAAAAAAGGTCTGGG

TCCTTGCAGACCACGCCCGACCAAGC

TTCGCCGGGCATGATCTGCAAGAACCCAG

ACCGGTGTTTCGTCCTTTCCACAA

Reverse antiMYCN-27

shRNA cloning primer.

Contains a BamHI-site

ON11 CGGGATCCAAAAAAGCTAGTGC

TCCTCGGCCTAGAAGGAGTAGCAAGC

TTCCCACTCCCTCCAGGCCGAGGAGCA

CCAGCGGTGTTTCGTCCTTTCCACAA

Reverse antiMYCN-1291

shRNA cloning primer.

Contains a BamHI-site

ON19 CGGGATCCAAAAAAGAATCACT

CAGAGTGTCCCCTCCGGAAGTGAAGCT

TGACCTCCGGAGAGGACACCCTGAGCG

ATTCGGTGTTTCGTCCTTTCCACAA

Reverse antiMYCN-760

shRNA cloning primer.

Contains a BamHI-site

ON20 CGGGATCCAAAAAAGTTCTTG

AGACACACAGCGATGGTAAATGGAAGCT

TGCATTCACCATCACTGTGCGTCCCAA

GAACGGTGTTTCGTCCTTTCCACAA

Reverse antiMYCN-887

shRNA cloning primer.

Contains a BamHI-site

ON22 ATAAGAATGCGGCCGCAAGGTC

GGGCAGGAAGAGGGCC

U6 forwar d primer.

Contains a NotI-site

ON51 CGGGATCCAAAAAAGAGCGTTCG

GAGCTGATGGCCATAAATACGAAGCTT

GGTACTTATGACCACCAACTCCGAA

CGCTCGGTGTTTCGTCCTTTCCACAA

Reverse cloning primer for

SCR shRNA.

Contains a BamHI-site

ON 56 ATTTGGGTCGCGGTTCTTG UBC forwar d QPCR primer

ON 57 TGCCTTGACATTCTCGATGGT UBC reverse QPCR primer

ON 87 CACCCTGAGCGATTCAGATGA MYCN forward QPCR

primer

ON 89 CCGGGACCCAGGGCT MYCN reverse QPCR primer

ON 58 GCAGCTACTCCTCCAGCTCT NFL forward QPCR primer

ON 59 ACTTGAGGTCGTTGCTGATG NFL reverse QPCR primer

ON 60 TCCAGCCCAGAGACACTGATT NPY forwar d QPCR primer

ON 61 AGGGTCTTCAAGCCGAGTTCT NPY reverse QPCR primer

ON 96 AAGTTCTACGGTGACGAGGAG CRT forward QPCR primer

ON 97 GTCGATGTTCTGCTCATGTTTC CRT reverse QPCR primer

ON 100 AGATCCCGGAGTTGGAAAAC c-MYC forward QPCR

primer

ON 101 AGCTTTTGCTCCTCTGCTTG c-MYC reverse QPCR primer

ON 170 TCACCCACACTGTGCCCATCTACGA β-actin forward QPCR primer

ON 171 CAGCGGAACCGCTCATTGCCAATGG β-actin reverse QPCR primer

ON 176 TGACACTGGCAAAACAATGCA HPRT1 forward QPCR

primer

192 Henriksen et al.

Name Sequence (5



-3



) Description

ON 177 GGTCCTTTTCACCAGCAAGCT HPRT1 reverse QPCR

primer

ON 304 CGAGAGCGAGCGGATGA CHGB forward QPCR

primer

ON 305 GGCGTGTCTTCACTTCTTCAGA CHGB reverse QPCR primer

2. Plasmids:

pSHAG-Ff1 (9) encodes an U6-driven anti-luciferase

(anti-luc) shRNA homologous to nucleotides 1,340–1,368

of the coding sequence of the ﬁreﬂy luciferase gene (NCBI

accession number U47296).

2.2. Cell Culture

and Transfection

1. Neuroblastoma cell line SK-N-BE(2) – MYCN ampliﬁed.

2. 6-well multiwell culture plates (Falcon).

3. RPMI-1640 supplemented with 10% fetal bovine serum

(FBS).

4. Phosphate-buffered saline (PBS).

5. Trypsin solution: 0.25% trypsin and 0.05% ethylenediamine

tetraacetic acid (EDTA) in PBS, pH 7.5.

6. Cells were maintained in a humidiﬁed 37

◦

C incubator with

5% CO

, supplied with fresh complete medium every 3 days

and subcultured before conﬂuence was reached.

7. Lipofectamin2000 (Invitrogen).

2.3. Cellular Protein

Isolation and

Western Blot

Analysis

1. Tropix lysis buffer, Protease inhibitor (Roche), DTT.

2. XCell SureLock

Mini-Cell (Invitrogen).

3. NuPAGE Novex 4–12% Bis-Tris Gel – 1.0 mm × 10 well

(Invitrogen).

4. NuPAGE LDS Sample Buffer (4×) (Invitrogen).

5. Molecular markers. MagicMark

XP western standard

(Invitrogen) as a protein size marker. Kaleidoscope

Prestained Standar d (Bio-RAD) for visualization of size

during electrophoresis and protein transfer efﬁciency from

gel to membrane during blotting.

6. Methanol.

7. Running buffer; NuPAGE MOPS SDS R unning Buffer

(20×) (Invitrogen).

8. XCell II

Blot Module (Invitr ogen).

9. Immobilon-FL PVDF transfer membrane (Millipore).

10. Whatman 3MM chromatography paper.

Inhibition of Gene Function in Mammalian Cells Using shRNA 193

11. Transfer buffer: NuPAGE Transfer Buffer (20×) (Invitro-

gen.)

12. Blocking buffer: Odyssey



Blocking buffer (LI-COR Bio-

sciences).

13. Primary Ab:

anti-N-myc mouse mAb (Calbiochem)

anti-actin rabbit pAb (Sigma)

14. Secondary Ab:

Alexa Fluor



680 conjugated goat anti-mouse IgG (Invit-

rogen)

IRDye800CW conjugated goat anti-rabbit IgG (Rockland)

15. PBST: PBS containing 0.1% Tween-20.

16. Odyssey Infrared Imaging System (LI-COR).

2.4. Total RNA

Isolation and cDNA

Synthesis

1. RNeasy Plus Mini Kit (Qiagen)

2. QIAshredder

(Qiagen)

3. Superscript

III reverse transcriptase (200 U/μL) (Invit-

rogen)

4. Oligo-dT

primer (50 μM)

5. dNTPs (2.5 mM each)

6. RNase inhibitor ( 20 U/μL)

7. Thermocycler

2.5. Real-Time

RT-PCR Analysis

1. Power SYBR Green PCR Master Mix (Applied Biosystems)

2. MicroAmp Optical 96-well Reaction Plate (Applied Biosys-

tems)

3. MicroAmp Optical Adhesive Film (Applied Biosystems)

4. 7,300 Real Time PCR System (Applied Biosystems)

5. 7,300 System Sequence Detection Software v1.4 (Applied

Biosystems)

6. qBase (http://medgen.ugent.be/qbase/)

3. Methods

This section gives a detailed description of the design and cloning

of anti-MYCN shRNA constructs. A detailed procedure for cell

transfection and evaluation of shRNA tr eated neuroblastoma

cells, using Wester n blot and real-time RT-PCR analysis, is also

described.