Human iPS RT-PCR Primer Set

Human iPS RT-PCR RT-PCR Primer Set, Complete set of RT-PCR primers for 43 marker genes for iPSC detection.

S

tem cells have two distinguishing features: features: 1. They have the ability to grow indenitely in an undifferentiated state and 2. They maintain pluripotency, the potential to develop into any type of cell. Embryonic stem (ES) cells were rst isolated from inner cell mass of mammalian mammalian blastocysts. 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. formation. They all form tightly packed and at colonies of cells, characterized by 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. RT-PCR. As demonstrated demonstrate d by Takahashi et al. in their landmark article in Cell , RT-PCR analysis of a set of ES cell markers should provide clear identication of stem cell colonies. In addition, RT-PCR RT-PCR was performed performed to demonstrate the presence of all three germ layers, or specic differentiation pathways.

Description The aim of Allele’s iPS RT-PCR RT-PCR primer sets is to provide researches with an efcient tools to initiate work in the iPS or ES eld. Primers are suitable for analyzing the reprogramming or differentiation stage of iPS or ES cells. Instead of ordering individual oligos, complete sets of primers, as precisely dened and tested in Takahashi Takahashi et al. 2007 [1], are conveniently and sufciently provided for 50 reactions for each gene. Each batch of primers is vigorously tested for oligo integrity. integrity.

Protocols RT-PCR: 1. Purify

total RNA from cells using RNA purication purication

kit or 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 manufacturer’s instructions. PCR protocol (using Allele-in-One Mix):

Box 2 | Recommended PCR Setup Component

Volume

Final Conc.

25µL

NA

Template DNA (1ng/µl)

1µl

20 pg/µl

Upstream primer (20

1 µl

0.6 µM

1 µl

0.6 µM

21 µl

NA

Allele-in One MasterMix

PCR

µM)

Downstream genespecic primer (20 µM) Nuclease-free water

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

Box 3 | Recommended PCR Setup

Features ♦ Pre-tested and complete set of RT-PCR RT-PCR primers for: 25 stem cell marker genes and 18 germ layer specic neuron or cardiomyocyte differentiation marker genes with housekeeping control. ♦ All sequences are identical to those as published by Takahashi et al. in their 2007 publication in Cell that Cell that demonstrated human iPS for the rst time.

Component

Volume

Final Conc.

Taq DNA Polymerase

5 units

NA

Template DNA (1ng/µl)

1µl

20 pg/µl

Upstream primer (20

1 µl

0.6 µM

µM)

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

Downstream genespecic primer (20 µM)

1 µl

0.6 µM

♦ Additional controls are included

10 X PCR buffer

5 µl

1X

10 mM dNTP mix

1 µl

Box 1 | Product Info Human iPS RT-PCR Primer Set CAT# ABP-SC-iPShRES

Nuclease-free water

0.2 mM

add to nal volume of 50 µl

Contents 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 g erm erm layer markers, 6 neuron markers, 4 cardiomyocute markers, 2 control house-keeping genes, as in [1]. Information is provided in Table 1-2 on the next page.

Perform 30 – 40 cycles of PCR amplication (2-step) as follows: Denature 94°C for 30 sec Anneal 58°C for 30 sec Anneal and extend 72°C for 1min

Allele Biotech-Introducing Cost Effectiveness to Research

References Takahashi, K., et al., Induction of pluripotent stem cells from adult human broblast by dened factors. Cell, 2007. 131(5): p. 861-72.

1.

FTherapeutic Use.

or Research Use Only. Not for Diagnostic or

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 Biotech is strictly prohibited

Table 1 | Human iPS RT-PCR Primers Symbol

Accession

Primers Sequence

Undifferentiated ES cell

OCT3/4 (POU5F1)

NM_002701.4

hOCT3/4-S1165:GACAGGGGGAGGGGAGGAGCTAGG


SOX2

NM_003106.2


NANOG


GDF3


ZFP42 (REX1)

NM_174900.3


FGF4

NM_002007.2


ESG1 (DPPA5)

NM_00102529 0.1

hpH34-S40:ATATCCCGCCGTGGGTGAAAGTTC


DPPA4

NM_018189.3

hECAT15-1-S532:GGAGCCGCCTGCCCTGGAAAATTC


DPPA2


TERT


KLF4


c-Myc


DNMT3B


GABRB3


TDGF1


GAL


LEFTY1 (LEFTB)

NM_020997.2


IFITM1

NM_003641.3


NODAL


UTF1


LEFTY2 (EBAF)

NM_003240.2


GRB7

NM_005310.2

Size

Applications

144 bp

Endo OCT3/4 RT-PCR

151 bp

Endo SOX2 RT-PCR

391 bp

NANOG RT-PCR

631 bp

GDF3 RT-PCR

306 bp

REX1 RT-PCR

371 bp

FGF4 RT-PCR

243 bp

ESG1/DPPA5 RT-PCR

408 bp

DPPA4 RT-PCR

608 bp

DPPA2 RT-PCR

446 bp

hTERT RT-PCR

397 bp

Endo KLF4 RT-PCR

328 bp

Endo c-MYC RT-PCR

242 bp

DNMT3B RT-PCR

277 bp

GABRB3 RT-PCR

237 bp

TDGF1 RT-PCR

188 bp

GAL RT-PCR

255 bp

LEFTB RT-PCR

229 bp

IFITM1 RT-PCR

234 bp

NODAL RT-PCR

171 bp

UTF1 RT-PCR

274 bp

EBAF RT-PCR

241 bp

GRB7 RT-PCR

hOCT3/4-AS1283:CTTCCCTCCAACCAGTTGCCCCAAAC hSOX2-S1430:GGGAAATGGGAGGGGTGCAAAAGAGG hSOX2-AS1555:TTGCGTGAGTGTGGATGGGATTGGTG NM_024865.2

ECAT4-macaca-968S:CAGCCC CGATTCTTCCACCAGTCCC ECAT4-macaca-1334AS: CGGAAGATTCCCAGTCGGGTTCACC

NM_020634.1

hGDF3-S243:CTTATGCTACGTAAAGGAGCTGGG hGDF3-AS850:GTGCCAACCCAGGTCCCGGAAGTT hREX1-RT-U:CAGATCCTAAACAGCTCGCAGAAT hREX1-RT-L:GCGTACGCAAATTAAAGTCCAGA hFGF4-RT-U:CTACAACGCCTACGAGTCCTACA hFGF4-RT-L:GTTGCACCAGAAAAGTCAGAGTTG

hpH34-AS259:ACTCAGCCATGGACTGGAGCATCC

hECAT15-1-AS916:TTTTTCCTGATATTCTATTCCCAT NM_138815.2

hECAT15-2-S85:CCGTCCCCGCAATCTCCTTCCATC hECAT15-2-AS667:ATGATGCCAACATGGCTCCCGGTG

NM_198253.2

hTERT-S3234:CCTGCTCAAGCTGACTCGACACCGTG hTERT-AS3713:GGAAAAGCTGGCCCTGGGGTGGAGC

NM_004235.4

hKLF4-AS1826:TGATTGTAGTGCTTTCTGGCTGGGCTCC hKLF4-S1128:ACGATCGTGGCCCCGGAAAAGGACC

NM_002467.3

hMYC-S253:GCGTCCTGGGAAGGGAGATCCGGAGC hMYC-AS555:TTGAGGGGCATCGTCGCGGGAGGCTG

NM_006892.3

hDNMT3B-S2502:TGCTGCTCACAGGGCCCGATACTTC hDNMT3B-S2716:TCCTTTCGAGC hDNMT3B-S2716:TCCTTTCGAGCTCAGTGCAC TCAGTGCACCACAAAAC CACAAAAC

NM_021912.3

hGABRB3-S1029:CCTTGCCCAAAATCCCCTATGTCAAAGC hGABRB3-AS1280:GTATCGCCAATGCCGCCTGAGACCTC

NM_003212.2

hTDGF1-S490:CTGCTGCCTGAATGGGGGAACCTGC hTDGF1-AS700:GCCACGAGGTGCTCATCCATCACAAGG

NM_015973.3

hGAL-S415:TGCGGCCCGAAGATGACATGAAACC hGAL-AS579:CCCAGGAGGCTCTCAGGACCGCTC hLEFTB-S794:CTTGGGGACTATGGAGCTCAGGGCGAC hLEFTB-AS1023:CATGGGCAGCGAGTCAGTCTCCGAGG hIFITM1-S166:CCCCAAAGCCAGAAGATGCACAAGGAG hIFITM1-AS368:CGTCGCCAACCATCTTCCTGTCCCTAG

NM_018055.4

hNODAL-S693:GGGCAAGAGGCACCGTCGACATCA hNODAL-AS900:GGGACTCGGTGGGGCTGGTAACGTTTC

NM_003577.2

hUTF1-S832:CCGTCGCTGAACACCGCCCTGCTG hUTF1-AS979:CGCGCTGCCCAGAATGAAGCCCAC hEBAF-S782:GCTGGAGCTGCACACCCTGGACCTCAG hEBAF-AS1032:GGGCAGCGAGGCAGTCTCCGAGGC hGRB7-S1250:CGCCTCTTCAAGTACGGGGTGCAGCTGT hGRB7-AS1467:TGGGCAGGCTGAGGCGGTGGTTTG


Table 2 | Human iPS RT-PCR Primers Undifferentiated

PODXL

NM_005397.3

ES cell Undifferentiated

CD9

NM_001769.2

hCD9-S369:GTGCATGCTGGGACTGTTCTTCGGCTTC

BXDC2

NM_018321.3

hBRIX-S596:CACCACGGTATCATCCCAAAAGCCAACC hBRIX-

220 bp

CD9 RT-PCR

230 bp

BRIX RT-PCR

307 bp

MSX1

AS798:ACGCCGATGCATGTTTGGTGACTGGTAG MSX1

NM_002448.3

hMSX1-S665:CGAGAGGACCCCGTGGATGCAGAG hMSX1-AS938:GGCGGCCATCTTCAGCTTCTCCAG

Mesoderm

PODXL RT-PCR

hCD9-AS564:CACGCCCCCAGCCAAACCACAGCAG

ES cell Mesoderm

226 bp

hPODXL-AS1403:CCGGGTTGAAGGTGGCTTTGACTGCTC

ES cell Undifferentiated

hPODXL-S1204:TCCAGCCCCACAGCAGCATCAACTACC

T, brachyury

NM_003181.2

homolog

hBRACHYURY-S1292:GCCCTCTCCCTCCCCTCCACGCACAG

RT-PCR 274 bp

hBRACHYURY-AS1540:CGGCGCCGTTGCTCACAGACCACAGG

BRACHYURY/T RT-PCR

(mouse) Ectoderm

PAX6

NM_001604.4

hPAX6-S1206:ACCCATTATCCAGATGTGTTTGCCCGAG

317 bp

hPAX6-AS1497:ATGGTGAAGCTGGGCATAGGCGGCAG Endoderm

FOXA2

NM_153675.2

hFOXA2-S208:TGGGAGCGGTGAAGATGGAAGGGCAC

RT-PCR 216 bp

hFOXA2-AS398:TCATGCCAGCGCCCACGTACGACGAC Endoderm

SOX17

NM_022454.3

hSOX17-S423:CGCTTTCATGGTGTGGGCTAAGGACG

AFP

NM_001134.1

hAFP-S948:GAATGCTGCAAACTGACCACGCTGGAAC

608 bp

KRT8(CK8)

NM_002273.3

hCK8-S734:CCTGGAAGGGCTGACCGACGAGATCAA hCK8-AS9

SOX17 RT-PCR

281 bp

hAFP-AS1201:TGGCATTCAAGAGGGTTTTCAGTCTGGA Endoderm

FOXA2 RT-PCR

hSOX17-AS583:TAGTTGGGGTGGTCCTGCATGTGCTG Endoderm

PAX6

AFP RT-PCR

247 bp

CK8 RT-PCR

233 bp

CK18 RT-PCR

212 bp

MAP2

56:CTTCCCAGCCAGGCTCTGCAGCTCC Endoderm

KRT18

NM_199187.1

(CK18) Ectoderm

MAP2

Neuron Marker Dopaminergic

GFAP

hCK18-S1125:AGCTCAACGGGATCCTGCTGCACCTTG hCK18AS1322:CACTATCCGGCGGGTGGTGGTCTTTTG

NM_00103953

hMAP2-S5401:CAGGTGGCGGACGTGTGAAAATTGAGAGTG

8.1

hMAP2-AS5587:CACGCTGGATCTGCCTGGGGACTGTG

NM_002055.3

hGFAP-S1040:GGCCCGCCACTTGCAGGAGTACCAGG hGFAP-

Neuron Markers

RT-PCR 328 bp

GFAP RT-PCR

242 bp

AADC RT-PCR

256 bp

ChAT RT-PCR

298 bp

SLC6A3/DAT

AS1342:CTTCTGCTCGGGCCCCTCATGAGACG

Dopaminergic

DDC

NM_00108297 hAADC-S1378:CGCCAGGATCCCCGCTTTGAAATCTG hAADC-

Neuron Markers

(AADC)

1.1

AS1594:TCGGCCGCCAGCTCTTTGATGTGTTC

Dopaminergic

CHAT

NM_020984.3

hChAT-S1360:GGAGGCGTGGAGCTCAGCGACACC hChAT-AS1

Neuron Markers

592:CGGGGAGCTCGCTGACGGAGTCTG

Dopaminergic

SLC6A3/

Neuron Markers

DAT DAT

Dopaminergic

LMX1B

NM_001044.3

hDAT-S1935:ACAGAGGGGAGGTGCGCCAGTTCACG hDAT-AS2 207:ACGGGGTGGACCTCGCTGCACAGATC

NM_002316.2

Neuron Markers

hLMX1B-S770:GGCACCAGCAGCAGCAGGAGCAGCAG

RT-PCR 276 bp

hLMX1B-AS1020:CCACGTCTGAGGAGCCGAGGAAGCAG

Cardiomyocyte

MYL7

NM_021223.2

hMYL2A-S258GGGCCCCATCAACTTCACCGTCTTCC hMYL2A-

Markers

(MYL2A)

Cardiomyocyte

TNNT2

NM_00100143

hTnTc-S524:ATGAGCGGGAGAAGGAGCGGCAGAAC hTnTc-

Markers

(TnTc)

2.1

AS730:TCAATGGCCAGCACCTTCCTCCTCTC

Cardiomyocyte

MEF2C

NM_002397.3

hMEF2C-S1407:TTTAACACCGCCAGCGCTCTTCACCTTG

PCR 236 bp

AS468TGTAGTCGATGTTCCCCGCCAGGTCC

Markers Myosi

Markers

(MYHCB)

NM_000257.2

hMYHCB-S5582:CTGGAGGCCGAGCAGAAGCGCAACG

MYL2A RTPCR

238 bp

TnTc RT-PCR

239 bp

MEF2C RT-

hMEF2C-AS1618:TCGTGGCGCGTGTGTTGTGGGTATCTCG

Cardiomyocyte

LMX1B RT-

PCR 258 bp

hMYHCB-AS5815:GTCCGCCCGCTCCTCTGCCTCATCC

MYHCB RTPCR

X : Signies corrections/typos to Takahashi, K., et al., Induction of pluripotent stem cells from adult human broblastsby dened factors. Cell, 2007. 131(5): p. 861-72.


Human iPS RT-PCR Primer Set

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