CAT# ABP-SC-iPShRESStorage conditions: -20 °C

BackgroundStem cells have two distinguishing features, 1) they have

and 2) they maintain pluripotency, the potential to develop into any type of cell.

cell mass of mammalian blastocysts. Induced pluripotent stem (iPS) cells are cells reprogrammed from adult cells.iPS cells resemble ES cells in morphology, proliferation, gene expression, and teratoma formation. They all form

large nuclei and scant cytoplasm [1]. Both types of stem cells can spontaneously differentiate or undergo induced or directed cell fate commitment.

One simple method for analyzing the differentiation status of iPS or ES cells is to use RT-PCR. As demonstrated by Takahashi et al. in their landmark article in Cell, RT-PCR analysis of a set of ES cell markers should provide clear

was performed to demonstrate the presence of all three

DescriptionThe aim of Allele’s iPS RT-PCR primer sets is to provide

for 50 reactions for each gene.

Features- Pre-tested, complete set of RT-PCR primers for 25 stem

or cardiomyocyte differentiation marker genes, with housekeeping control.

- All sequences are identical to those as published by Takahashi et al. in their 2007’s publication in Cell that

- All oligos were produced in-house at Allele Biotech with quality control--provided at uniform concentrations for easy reaction setup.

- Additional controls are included.

ApplicationsSuitable for analyzing the reprogramming or differentiation stage of iPS or ES cells.

Quality AssuranceEach batch of primers is vigorously tested for oligo integraty. The following regents are provided in the kit:

-- dT(20) primer for reverse transcription (50 µl at 20 µM)

-- 45 pairs of RT-PCR primers

Primers are provided for 25 ES marker genes, 8 derm layer markers, 6 neuron markers, 4 cardiomyocute markers, 2 control house-keeping genes, as in [1]. Information is provided in Table 1 on the next page.

Recommended ProtocolsRT-PCR:

reagent.

2) Use 1 µg total RNA and 1 µl of 20 µM dT(20) in reserve transcription reaction with a high quality reverse transcriptase. Follow manufacturer’s instructions

PCR protocol (using Allele-in-One Mix):

Component Volume Final ConcentrationTemplate DNA (1ng/µl) 1µl 20 pg/µlUpstream primer (20 µM) 1 µl 0.6 µMDownstream gene-

1 µl 0.6 µMDistilled water 21 µlAllele-in-one Mix 25 µl

The following procedure is suggested as a starting point when using Taq polymerase:

Component Volume Final ConcentrationTemplate DNA (1ng/µl) 1 µl 20 pg/µl10 X PCR buffer 5 µl 1X10 mM dNTP mix 1 µl 0.2 mMUpstream primer (20 µM) 1 µl 0.6 µMDownstream gene-

1 µl 0.6 µMTaq DNA Polymerase 5 unitDistilled water

follows:Denature 94°C for 30 secAnneal 58°C for 30 secAnneal and extend 72°C for 1min

1. Takahashi, K., et al., Induction of pluripotent stem cells from adult

For Research Use Only. Not for Diagnostic or Therapeutic Use.

Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Allele, Inc. is strictly prohibited.

iPS RT-PCRiPS RT-PCRPrimer SetPrimer Set 9924 Mesa Rim Road. San Diego, CA 92121

tel: 858-587-6645, 800-991-RNAi fax: 858-587-6692 website: www.allelebiotech.com email: oligo@allelebiotech.com

TABLE 1: Human iPS RT-PCR PrimersTABLE 1: Human iPS RT-PCR PrimersSymbol Accession Primers Size Applications

Undifferentiated

ES cell

OCT3/4

(POU5F1)

NM_002701.4 hOCT3/4-S1165:GACAGGGGGAGGGGAGGAGCTAGG

hOCT3/4-AS1283:CTTCCCTCCAACCAGTTGCCCCAAAC

144

bp

Endo OCT3/4

RT-PCR

Undifferentiated

ES cell

SOX2 NM_003106.2 hSOX2-S1430:GGGAAATGGGAGGGGTGCAAAAGAGG

hSOX2-AS1555:TTGCGTGAGTGTGGATGGGATTGGTG

151

bp

Endo SOX2

RT-PCR

Undifferentiated

ES cell

NANOG NM_024865.2 ECAT4-macaca-968S:CAGCCCCGATTCTTCCACCAGTCCC

ECAT4-macaca-1334AS:CGGAAGATTCCCAGTCGGGTTCACC

391

bp

NANOG

RT-PCR

Undifferentiated

ES cell

GDF3 NM_020634.1 hGDF3-S243:CTTATGCTACGTAAAGGAGCTGGG

hGDF3-AS850:GTGCCAACCCAGGTCCCGGAAGTT

631

bp

GDF3

RT-PCR

Undifferentiated

ES cell

ZFP42 (REX1) NM_174900.3 hREX1-RT-U:CAGATCCTAAACAGCTCGCAGAAT

hREX1-RT-L:GCGTACGCAAATTAAAGTCCAGA

306

bp

REX1

RT-PCR

Undifferentiated

ES cell

FGF4 NM_002007.2 hFGF4-RT-U:CTACAACGCCTACGAGTCCTACA

hFGF4-RT-L:GTTGCACCAGAAAAGTCAGAGTTG

371

bp

FGF4

RT-PCR

Undifferentiated

ES cell

ESG1 (DPPA5) NM_00102529

0.1

hpH34-S40:ATATCCCGCCGTGGGTGAAAGTTC

hpH34-AS259:ACTCAGCCATGGACTGGAGCATCC

243

bp

ESG1/DPPA5

RT-PCR

Undifferentiated

ES cell

DPPA4 NM_018189.3 hECAT15-1-S532:GGAGCCGCCTGCCCTGGAAAATTC

hECAT15-1-AS916:TTTTTCCTGATATTCTATTCCCAT

408

bp

DPPA4

RT-PCR

Undifferentiated

ES cell

DPPA2 NM_138815.2 hECAT15-2-S85:CCGTCCCCGCAATCTCCTTCCATC

hECAT15-2-AS667:ATGATGCCAACATGGCTCCCGGTG

608

bp

DPPA2

RT-PCR

Undifferentiated

ES cell

TERT NM_198253.2 hTERT-S3234:CCTGCTCAAGCTGACTCGACACCGTG

hTERT-AS3713:GGAAAAGCTGGCCCTGGGGTGGAGC

446

bp

hTERT

RT-PCR

Undifferentiated

ES cell

KLF4 NM_004235.4 hKLF4-AS1826:TGATTGTAGTGCTTTCTGGCTGGGCTCC

hKLF4-S1128:ACGATCGTGGCCCCGGAAAAGGACC

397

bp

Endo KLF4

RT-PCR

Undifferentiated

ES cell

c-Myc NM_002467.3 hMYC-S253:GCGTCCTGGGAAGGGAGATCCGGAGC

hMYC-AS555:TTGAGGGGCATCGTCGCGGGAGGCTG

328

bp

Endo c-MYC

RT-PCR

Undifferentiated

ES cell

DNMT3B NM_006892.3 hDNMT3B-S2502:TGCTGCTCACAGGGCCCGATACTTC

hDNMT3B-S2716:TCCTTTCGAGCTCAGTGCACCACAAAAC

242

bp

DNMT3B

RT-PCR

Undifferentiated

ES cell

GABRB3 NM_021912.3 hGABRB3-S1029:CCTTGCCCAAAATCCCCTATGTCAAAGC

hGABRB3-AS1280:GTATCGCCAATGCCGCCTGAGACCTC

277

bp

GABRB3

RT-PCR

Undifferentiated

ES cell

TDGF1 NM_003212.2 hTDGF1-S490:CTGCTGCCTGAATGGGGGAACCTGC

hTDGF1-AS700:GCCACGAGGTGCTCATCCATCACAAGG

237

bp

TDGF1

RT-PCR

Undifferentiated

ES cell

GAL NM_015973.3 hGAL-S415:TGCGGCCCGAAGATGACATGAAACC

hGAL-AS579:CCCAGGAGGCTCTCAGGACCGCTC

188

bp

GAL

RT-PCR

Undifferentiated

ES cell

LEFTY1

(LEFTB)

NM_020997.2 hLEFTB-S794:CTTGGGGACTATGGAGCTCAGGGCGAC

hLEFTB-AS1023:CATGGGCAGCGAGTCAGTCTCCGAGG

255

bp

LEFTB

RT-PCR

Undifferentiated

ES cell

IFITM1 NM_003641.3 hIFITM1-S166:CCCCAAAGCCAGAAGATGCACAAGGAG

hIFITM1-AS368:CGTCGCCAACCATCTTCCTGTCCCTAG

229

bp

IFITM1

RT-PCR

Undifferentiated

ES cell

NODAL NM_018055.4 hNODAL-S693:GGGCAAGAGGCACCGTCGACATCA

hNODAL-AS900:GGGACTCGGTGGGGCTGGTAACGTTTC

234

bp

NODAL

RT-PCR

Undifferentiated

ES cell

UTF1 NM_003577.2 hUTF1-S832:CCGTCGCTGAACACCGCCCTGCTG

hUTF1-AS979:CGCGCTGCCCAGAATGAAGCCCAC

171

bp

UTF1

RT-PCR

Undifferentiated

ES cell

LEFTY2 (EBAF) NM_003240.2 hEBAF-S782:GCTGGAGCTGCACACCCTGGACCTCAG

hEBAF-AS1032:GGGCAGCGAGGCAGTCTCCGAGGC

274

bp

EBAF

RT-PCR

Undifferentiated

ES cell

GRB7 NM_005310.2 hGRB7-S1250:CGCCTCTTCAAGTACGGGGTGCAGCTGT

hGRB7-AS1467:TGGGCAGGCTGAGGCGGTGGTTTG

241

bp

GRB7

RT-PCR

Undifferentiated

ES cell

PODXL NM_005397.3 hPODXL-S1204:TCCAGCCCCACAGCAGCATCAACTACC

hPODXL-AS1403:CCGGGTTGAAGGTGGCTTTGACTGCTC

226

bp

PODXL

RT-PCR

Undifferentiated

ES cell

CD9 NM_001769.2 hCD9-S369:GTGCATGCTGGGACTGTTCTTCGGCTTC

hCD9-AS564:CACGCCCCCAGCCAAACCACAGCAG

220

bp

CD9

RT-PCR

Undifferentiated

ES cell

BXDC2 NM_018321.3 hBRIX-S596:CACCACGGTATCATCCCAAAAGCCAACC

hBRIX-AS798:ACGCCGATGCATGTTTGGTGACTGGTAG

230

bp

BRIX

RT-PCR

Mesoderm MSX1 NM_002448.3 hMSX1-S665:CGAGAGGACCCCGTGGATGCAGAG

hMSX1-AS938:GGCGGCCATCTTCAGCTTCTCCAG

307

bp

MSX1

RT-PCR

Mesoderm T, brachyury

homolog (mouse)

NM_003181.2 hBRACHYURY-S1292:GCCCTCTCCCTCCCCTCCACGCACAG

hBRACHYURY-AS1540:CGGCGCCGTTGCTCACAGACCACAGG

274

bp

BRACHYURY/T

RT-PCR

Ectoderm PAX6 NM_001604.4 hPAX6-S1206:ACCCATTATCCAGATGTGTTTGCCCGAG

hPAX6-AS1497:ATGGTGAAGCTGGGCATAGGCGGCAG

317

bp

PAX6

RT-PCR

Endoderm FOXA2 NM_153675.2 hFOXA2-S208:TGGGAGCGGTGAAGATGGAAGGGCAC

hFOXA2-AS398:TCATGCCAGCGCCCACGTACGACGAC

216

bp

FOXA2

RT-PCR

Endoderm SOX17 NM_022454.3 hSOX17-S423:CGCTTTCATGGTGTGGGCTAAGGACG

hSOX17-AS583:TAGTTGGGGTGGTCCTGCATGTGCTG

608

bp

SOX17

RT-PCR

Endoderm AFP NM_001134.1 hAFP-S948:GAATGCTGCAAACTGACCACGCTGGAAC

hAFP-AS1201:TGGCATTCAAGAGGGTTTTCAGTCTGGA

281

bp

AFP

RT-PCR

Endoderm KRT8(CK8) NM_002273.3 hCK8-S734:CCTGGAAGGGCTGACCGACGAGATCAA

hCK8-AS956:CTTCCCAGCCAGGCTCTGCAGCTCC

247

bp

CK8

RT-PCR

Endoderm KRT18 (CK18) NM_199187.1 hCK18-S1125:AGCTCAACGGGATCCTGCTGCACCTTG

hCK18-AS1322:CACTATCCGGCGGGTGGTGGTCTTTTG

233

bp

CK18

RT-PCR

Ectoderm

Neuron Marker

MAP2 NM_00103953

8.1

hMAP2-S5401:CAGGTGGCGGACGTGTGAAAATTGAGAGTG

hMAP2-AS5587:CACGCTGGATCTGCCTGGGGACTGTG

212

bp

MAP2

RT-PCR

Dopaminergic

Neuron Markers

GFAP NM_002055.3 hGFAP-S1040:GGCCCGCCACTTGCAGGAGTACCAGG

hGFAP-AS1342:CTTCTGCTCGGGCCCCTCATGAGACG

328

bp

GFAP

RT-PCR

Dopaminergic

Neuron Markers

DDC（AADC） NM_00108297

1.1

hAADC-S1378:CGCCAGGATCCCCGCTTTGAAATCTG

hAADC-AS1594:TCGGCCGCCAGCTCTTTGATGTGTTC

242

bp

AADC

RT-PCR

Dopaminergic

Neuron Markers

CHAT NM_020984.3 hChAT-S1360:GGAGGCGTGGAGCTCAGCGACACC

hChAT-AS1592:CGGGGAGCTCGCTGACGGAGTCTG

256

bp

ChAT

RT-PCR

Dopaminergic

Neuron Markers

SLC6A3/DAT NM_001044.3 hDAT-S1935:ACAGAGGGGAGGTGCGCCAGTTCACG

hDAT-AS2207:ACGGGGTGGACCTCGCTGCACAGATC

298

bp

SLC6A3/DAT

RT-PCR

Dopaminergic

Neuron Markers

LMX1B NM_002316.2 hLMX1B-S770:GGCACCAGCAGCAGCAGGAGCAGCAG

hLMX1B-AS1020:CCACGTCTGAGGAGCCGAGGAAGCAG

276

bp

LMX1B

RT-PCR

Cardiomyocyte

Markers

MYL7(MYL2A) NM_021223.2 hMYL2A-S258GGGCCCCATCAACTTCACCGTCTTCC

hMYL2A-AS468TGTAGTCGATGTTCCCCGCCAGGTCC

236

bp

MYL2A

RT-PCR

Cardiomyocyte

Markers

TNNT2（TnTc） NM_00100143

2.1

hTnTc-S524:ATGAGCGGGAGAAGGAGCGGCAGAAC

hTnTc-AS730:TCAATGGCCAGCACCTTCCTCCTCTC

238

bp

TnTc

RT-PCR

Cardiomyocyte

Markers

MEF2C NM_002397.3 hMEF2C-S1407:TTTAACACCGCCAGCGCTCTTCACCTTG

hMEF2C-AS1618:TCGTGGCGCGTGTGTTGTGGGTATCTCG

239

bp

MEF2C

RT-PCR

Cardiomyocyte

Markers

Myosi

(MYHCB)

NM_000257.2 hMYHCB-S5582:CTGGAGGCCGAGCAGAAGCGCAACG

hMYHCB-AS5815:GTCCGCCCGCTCCTCTGCCTCATCC

258

bp

MYHCB

RT-PCR

X: Signifies corrections/typos to Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblastsby defined factors.

Cell, 2007. 131(5): p. 861-72.