Nielsen H. RNA: Methods and Protocols

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324 Nielsen

cell RNA is in the range 1–4% dependent on cell type. Thus, less

than 100 ng of whole cell RNA should be sufﬁcient to produce

the 1 ng of dsDNA required as the input material for a sequenc-

ing run. Given that a typical mammalian cell contains 10–30 pg of

RNA this corresponds to a few thousand cells. However, this is a

naïve calculation as transcripts vary several orders of magnitude in

abundance and, as a consequence, much more RNA is required

to obtain enough sequence depth to quantitate low abundance

transcripts. Many biological questions require that fewer cells are

being analyzed and this appears to be the goal of new develop-

ments, e.g., by direct sequencing of RNA molecules (2).

It is important to prevent loss of material due to adsorption

to surfaces when handling small amounts of nucleic acid. “Non-

stick” (e.g., siliconized or Teﬂon-coated) disposable plasticware

should be used and detergent (e.g., 0.02% Tween-20) included

in reaction steps. In precipitation reactions, glycogen should be

included to facilitate recovery and handling of the nucleic acid. A

convenient way of shipping nucleic acids is to perform an ethanol

precipitation, remove most of the ethanol by aspiration, and leave

the material as a wet (ethanol) pellet (dry pellets will not stay at

the bottom of the tube). Then, the tube is wrapped in paraﬁlm

and shipped. DNA can be shipped at ambient temperature and

this is also possible with RNA for some applications.

2.3. Preparation of

RNA for miRNA

Proﬁling

Transcriptome analysis of the miRNA fraction of RNA or

“miRNA proﬁling” is an important type of analysis in biology

and medicine. The principal methods are qRT-PCR, microar-

ray hybridization, and deep sequencing. A very useful reference

for comparison of the three methods is Git et al. (7). In this

paper three biological samples were analyzed on six different

microarray platforms and by sequencing, and 89 miRNA wer e

further validated by qRT-PCR. The study discloses the strengths

and weaknesses of each of the methods and provides an excel-

lent example of the difﬁculties in dealing with genome-wide

datasets.

The steps in preparation of samples for miRNA proﬁling gen-

erally involves isolation of whole cell RNA using a variation of the

acidic phenol/guanidinium thiocyanate method (6) followed by

size-fractionation to obtain the small RNA (<200 nt) fraction.

There are several methods for this, e.g., denaturing polyacry-

lamide gel electrophoresis or differential elution from an RNA-

binding (silica disc) matrix.

miRNA proﬁling typically starts with 1–10 μg of whole cell

RNA. The low molecular weight fraction that is isolated is typ-

ically 10–15%. Most of this is 5S rRNA, 5.8S rRNA, and tRNA

and the miRNA constitutes considerably less than 1%.

The comments on handling and shipping of samples are sim-

ilar to those described in Section 2.2.

Outsourcing of Experimental Work 325

2.4. Preparation of

Protein Samples

Protein identiﬁcation, e.g., of an RNA-binding protein by mass

spectrometry (“mass-spec” or MS) is relatively straightforward

and is typically done as a service by a core facility or commer-

cial service. These services have been around much longer than

facilities handling nucleic acids for the purposes described above

and sample preparation and data handling are quite simple. Gen-

eral proteomic analysis and characterization of proteins with car-

bohydrate, lipid, or other modiﬁcations is of course a different

story, but this is beyond the scope of this chapter. The amount

of protein required for identiﬁcation is typically 50–100 ng. In

order to obtain this amount for a 50-kDa protein found in 1,000

copies per cell, 20 mg of total protein from 1.2 × 10

cells are

required as starting material provided 100% efﬁciency of all pro-

cedures used. The protein is provided as a stained gel band or

spot stained with a MS-compatible stain. Coomassie blue stain

has a detection limit (100 ng) corresponding to the amount

required for MS-analysis and is a good choice. Tips for sam-

ple preparation can be found at the homepages of MS-services,

e.g., http://www.albany.edu/genomics/proteomics-sample-

preparation.html.

Reference

1. Metzker, M. L. (2010) Sequencing technolo-

gies – the next generation. Nat Rev Genet 11,

31–46.

2. Ozsolak, F., Platt, A. R., Jones, D. R., Reifen-

berger,J.G.,Sass,L.E.,McInerney,P.,

Thompson, J. F., Bowers, J., Jarosz, M.,

Milos, P. M. (2009) Direct RNA sequencing.

Nature 461, 814–818.

3. McPherson, J. D. (2009) Next-generation

gap. Nat Methods 6, S2–S5.

4. Linnarsson, S. (2010) Recent advances in

DNA sequencing methods – general princi-

ples of sample preparation. Exp Cell Res 316,

1339–1343.

5. Mortazavi, A., Williams, B. A., McCue,

K., Schaeffer, L., Wold, B. (2008) Map-

ping and quantifying mammalian tran-

scriptomes by RNA-Seq. Nat Methods 5,

621–628.

6. Chomczynski, P., Sacchi, N. (1987)

Single-step method of RNA isolation

by acid guanidinium thiocyanate-phenol-

chloroform extraction. Anal Biochem 162,

156–159.

7. Git, A., Dvinge, H., Salmon-Divon, M.,

Osborne, M., Kutter, C., Hadﬁeld, J.,

Bertone, P., Caldas, C. (2010) Systematic

comparison of microarray proﬁling, real-

time PCR, and next-generation sequenc-

ing technologies for measuring differ-

ential microRNA expression. RNA 16,

991–1006.

SUBJECT INDEX

ActinomycinD......................................6

Agarose gel electrophoresis

formaldehyde...........25, 89–90, 95–96, 98–99, 256

glyoxal.....................89–90, 96, 100, 103–104

TAE/formamide...........................297, 304

Alkaline Phosphatase

Calfintestinalphosphatase(CIP)............278, 281

Shrimp alkaline phosphatase (SAP) . 108, 110, 113–114

Alternativesplicing...............12, 137–138, 140–148,

151, 161, 164, 239–240, 242

Antisense . . . 6, 8, 10–11, 29, 111, 117, 173–176, 189–190,

195, 206–207, 238–

239, 312

Argonaute...........................174–175, 190, 240

Atomicforcemicroscopy(ATM)...........141, 266–267

Autoradiography..................37, 102, 162–163, 169

Basic local alignment search tool (BLAST) . 54, 69–70, 73,

184, 194, 315

Biotin......................................48–50, 125

BLAST-likealignmenttool(BLAT)............. 54, 141

Caenorhabditis elegans .........47, 144, 147, 173–175, 252

Cap (m

Gcap)..........................7, 33, 108, 293

Capanalogue..............................33, 128, 130

Cap analysis of gene expression (CAGE) .............7–8

Capillaryblotting ............................ 91, 98–99

cDNA synthesis. . . .193, 198–199, 207, 209–210, 257, 323

Cerenkovcounting............................281, 288

Chelex...........................16, 222–224, 227–229

Chromatin immunoprecipitation (ChIP) . . . . 219–233, 320

Co-precipitant

glycogen...............................47, 288, 324

linearacrylamide.................................23

tRNA.................................23, 273, 288

Covariancemodel.....................70, 74–75, 77, 80

Cross-linking and immunoprecipitation

(CLIP-Chip).......................242–243

Crosslinking..............17, 26, 222, 226, 242

, 260, 287

Crypticunstabletranscript(CUT)................ 8, 238

Deep sequencing . . . . . . . 4, 8, 219, 222, 242, 249, 320, 324

Dicer ...................... 174–175, 180–181, 190, 206

Diethylpyrocarbonate (DEPC) . . 17, 26–27, 37, 39, 95–97,

111, 115, 134, 168, 251, 258–259, 279, 303, 305

DNApolymerase..................23, 35, 150–151, 153

DNaseI ................................. 111, 115, 170

Dot/slotblot...................................43, 222

Double-stranded RNA

(dsRNA).........173–174, 177–181, 190, 207

DpnI................................150–151, 153

, 155

Drosha...................................180–181, 190

Drosophila melanogaster .............................252

Electroblotting..................................91–92

Electroelution.......................................22

Electrophoretic mobility shift assay (EMSA) . . . . . 275–290

ENcyclopedia of DNA elements

(ENCODE)............ 5, 9, 63, 69, 239, 321

Escherichia coli ......................23, 29, 40, 128, 142,

150, 153, 208, 251, 288

Ethidium bromide

(EtBr). . 26, 36–37, 90–92, 95, 98–99, 101–102,

113–114, 119, 299, 304, 307, 322

Expressed sequence tag (EST) . . . . . . . . . . . . 6, 43, 141–145

FASTAformat......................................72

FLAGtag................................249, 266–272

Flprecombinase...............................269–270

Formaldehyde...............25, 89–90, 95, 98, 103–104,

222, 224–226, 230–232, 256, 260, 295

Gelﬁltration..........................18, 21, 34–35, 37

Gelpuriﬁcation...........18, 21–22, 34, 36–37, 281–282

Gel-shift.................................275–277, 286

Genesilencing .........174–177, 181–184, 189–190, 207

Genome browser

Ensembl.................11, 53–54, 56–65, 143–146

NCBI..........................53–54, 69, 192, 194

UCSC............................11, 53–59, 62–64

Guanidinium thiocyanate (GuSCN, GuITC or

GTC)........17, 20, 24, 44–45, 47–48, 50–51,

88, 125, 128, 131–132, 134, 299, 303–304,

323–324

Guidestrand.........................174–175, 185, 190

HeLacell............................207–208, 213, 306

Helicase......................................177, 190

Heparin..........................17, 295, 298–299, 307

hnRNP...........................................139

Housekeeping gene . . . . . . . . . . . . . . . . . . . 202–204, 213, 216

Humancells................8, 47, 88, 174, 177, 184, 239

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

DOI 10.1007/978-1-59745-248-9,

 Springer Science+Business Media, LLC 2011

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328

RNA

Subject Index

Hybridization . . . 4, 29, 33–34, 50, 87–94, 97–98, 102–104,

162, 166–170, 222, 257, 315, 319, 324

ImageQuantsoftware..........................285–286

Immunoprecipitation.........63, 219–233, 242, 247–260,

266–267, 272–273

Innateimmunity.....................175, 178–181, 183

Invitrtranscription..............29–40, 88, 93, 108, 112,

129–130, 257

Labelling.............23, 33, 93, 103, 165, 167–168, 170

Lockednucleicacid(LNA)..................43–51, 189

Luciferase.......................192, 207–208, 213–214

Lyophilization.....................................280

Lysis.........17, 44–46, 50, 192, 196, 203, 222, 224–228,

231, 233, 250, 252, 259, 266, 268, 271–273,

295, 300, 250, 252, 259, 266, 268, 271–273,

295, 300, 306

Magneticbead.......................251, 253–254, 256

Magneticparticles........................45–47, 49, 51

See also Magnetic bead

Passenger strand . . . . . . . . . . . . . . . . . . . . . . . . . . 175–176, 190

Peptidenucleicacid(PNA)......................44, 189

Phenol:chloroform:isoamylalcohol (PCI) . . . . . . . 19, 27, 34

Phenol extraction (phenol/chloroform extraction) . 20, 129,

222, 280, 288

Phosphoimager...........................131, 133, 169

PlacentalRNaseinhibitor............................26

Polyacrylamide gel electrophoresis

denaturing(urea)...................26, 100, 166, 169

native.............................48, 276–277, 283

SDS-PAGE.......................... 260, 266, 271

Polyadenylation.......9–11, 126, 128, 130–131, 134, 138,

130–131, 134, 138, 239, 241–243

Poly(A)

mRNA ................................ 19–20

Poly(A)polymerase(E-PAP).....126, 128, 130–131, 134

Polymerasechainreaction(PCR)...........108, 205–217

Polynucleotide kinase (T4 PNK) . . . 23, 165, 167–168, 278,

281

Polysomeproﬁling...............123, 294–298, 303–307

Polysomerun-off..............................294–295

Post-transcriptionalgenesilencing(PTGS)......174, 189

Post-transcriptionalmodiﬁcation................237, 246

Post-transcriptionaloperon(PTO).....237–244, 248, 266

Post-translationalmodiﬁcation.............138, 239, 248

Primer3program...........................39, 121, 216

Probelabelling.......................................4

Promoter . . 4, 6–10, 29–32, 34–36, 39, 120, 137, 150, 156,

195–196, 220, 238–239, 267

PROMPTS....................................7–8, 10

Proteinase K . . 20, 34, 36, 44, 111, 115, 150, 153, 223–224,

227, 229, 250, 252, 255, 260

Quantitative reverse transcription-PCR

(qRT-PCR) . . . . 48–49, 193, 200–202, 295, 324

RadiolabeledRNA........................276–282, 289

Randomhexamerprimers......................203, 323

Rapid ampliﬁcation of cDNA ends (RACE) . . . . . 107–121

Reporterconstruct.............................142, 153

Reversetranscriptase..........6, 108–109, 111, 117, 151,

162, 193, 198, 151, 162, 193, 198, 203,

208–209, 307

Reverse transcr iption-PCR

(RT-PCR) . . . . . . 48–49, 94, 108, 150, 153–154,

161–162, 193, 200–202, 250, 256, 304

Rfamdatabase......................................77

RiboGreen.........................................19

Ribonome.........................................241

Ribonomicproﬁling.......................249–251, 258

Ribonucleoprotein(RNP)........181, 237–244

, 247–260,

265–273, 275–290, 293–307, 311–316,

319–325

Ribosomal RNA (rRNA) . . . . . 3, 11, 15–16, 23, 25–26, 67,

25–26, 67, 70, 90–91, 98, 256, 268, 273, 324

Riboswitch..............................12, 67, 71, 149

Ribozyme.................12, 29, 32, 34, 67, 70, 74, 189

RNA-binding protein immunoprecipitation-microchip

proﬁling (RIP-Chip) . . . 242–244, 247–260, 266

RNA binding protein (RBP) . . 29, 237

, 241–243, 247–260,

265–272, 286–287, 289, 320, 325

RNAfolding............18, 24–25, 27, 32, 73, 81–83, 89

RNA-induced silencing complex (RISC) . . . . . . . . 174–176,

190, 207

RNA interf erence (RNAi) . . . . . . . 173–185, 190, 205–217,

293–294

RNA isolation. . . . .18, 44, 47, 50, 193, 198, 207, 209, 299,

303–304

RNAmotif.......16, 67–69, 71–74, 80, 83, 148–

149, 179

RNA

Subject Index

329

RNA polymerase II (RNAP II, pol II) . . . 5–6, 8, 180, 190,

195, 219–233, 239

RNApolymeraseIII...........................190, 195

RNA precipitation

ethanol.....................................24, 207

isopropanol................................132–133

LiCl....................................22–23, 307

RNAquantitation..................................162

RNAregulon......................................241

RNase.................12, 16–18, 21, 23, 26–27, 29, 36,

43–45, 95, 99, 104, 110–111, 115–116,

127–128, 130, 134, 168, 170, 190, 193, 198,

203, 206, 127

–128, 130, 134, 168, 170, 190,

193, 198, 203, 206, 190, 193, 198, 203, 206,

257–258, 268, 278–280, 290, 295, 303–306

RNaseH.................................111, 117, 124

RNase inhibitor17, 26–27, 44, 193, 198, 252–253, 268, 306

RNaseprotection.......................17, 29, 257, 295

RNAstability.................................243, 266

RNA structure . . . . . 5, 12, 17, 24, 29, 67–83,

181, 190, 194

RNPgranule..................................265–273

Saccharomyces cerevisiae .........................265, 268

Secondarystructure .................25, 70–83, 89, 142,

148, 162, 148, 162, 170, 176–177, 179, 194,

206, 278

Seedsequence............................175, 312, 314

Sephacryl.......................................21, 35

Sephadex....................21, 128, 130, 165, 168, 278

Sepharose..................223, 251, 253–254, 256, 259

Sequencealignment.........60, 68, 70, 73, 75–78, 81, 83

Serial analysis of gene expression (SAGE) . . . . . . . 242, 249

Short hairpin RNA (shRNA). . . . .156, 180, 189–204, 207

213–214

Single nucleotide polymorphism (SNP) . . . . . 140, 142, 148

Single-stranded RNA (ssRNA). . . . . . 18, 77, 178–179, 206

Small interfering RNA (siRNA) . . 150, 174–185, 189–190,

194, 207

SmallnuclearRNA(snRNA)....................67, 100

Small nucleolar RNA (snoRNA). . . . . . . 10–11, 67, 70, 100

S1nuclease...................................161–171

Sonication......................222–228, 231–233, 271

Southernblot......................87, 91, 119, 270, 272

Splicing

splice site . . 138–140, 142, 145, 147–149, 152, 166, 169

splicingenhancer...............................139

splicingsilencer.................................139

SRproteins........................................139

Streptavidin...............44–47

, 50, 124–125, 128, 132

Sucrosegradient...............................293–307

SYBR Green . . 193, 199, 205–206, 208, 210, 224, 229, 322

Targetknockdownvalidation...................175, 189

Templatepuriﬁcation..........................127, 129

Tetracycline...................................266–271

Thin-layerchromatography(TLC)...................21

Tilingarray...................................4, 8, 257

Tobacco acid pyrophosphatase (TAP) . . 108, 110–111, 114,

146, 249

Toll-like receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

Transcriptioninitiation.......4, 7–8, 11, 33, 88, 109, 239

Transcriptioninterference.....................6, 10, 238

Transcriptionstartsite(TSS)....7–11, 108, 138, 220, 239

Transcriptiontermination.....................9, 31, 195

Transcriptionunit............3–4, 8–12, 53–54, 238–239

Transfection . . 150–151, 153–156, 182,

192–193, 196, 200,

208–209, 216, 268–270, 272

Tr ansfer RNA (tRNA) . . . . 3, 15–16, 23, 26, 32, 45–46, 32,

45–46, 67, 70–71, 129, 133, 166, 273, 283–284,

287–289, 293–295, 324

Translationalcontrol ............................... 293

Trizol......................170, 207, 209, 221, 273, 307

T4RNAligase.......................108, 111, 116, 120

T7RNApolymerase.............30, 33, 35, 37, 180

, 277

Untranslatedregion(UTR)...........6, 9–10, 55–58, 65,

68–69, 71, 82–83, 120, 125, 147, 149, 239–240,

242–244, 248, 256, 294, 312, 242–244, 248,

256, 294, 312, 314

UV-shadowing......................................21

UV-spectroscopy...........................18–19, 207

Western blot . . . . 87, 92, 192–193, 260, 266–267, 270, 307

Xenopus laevis ......................................134