Human iPS RT-PCR Primer Set, Complete set of RT-PCR

primers for 43 marker genes for iPSC detection.

Protocols
RT-PCR: 1. Purify total RNA from cells using RNA purification kit or reagent. Use 1 g total RNA and 1 l of 20 M dT(20) in 2. reserve transcription reaction with a high quality reverse transcriptase. Follow manufacturers instructions. PCR protocol (using Allele-in-One Mix):

tem cells have two distinguishing features: 1. They have the ability to grow indefinitely in an undifferentiated state and 2. They maintain pluripotency, the potential to develop into any type of cell. Embryonic stem (ES) cells were first isolated from inner 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 tightly packed and flat 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. 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 identification of stem cell colonies. In addition, RT-PCR was performed to demonstrate the presence of all three germ layers, or specific differentiation pathways.

Box 2 | Recommended PCR Setup

Component Allele-in One MasterMix PCR Volume 25L 1l 1 l 1 l 21 l Final Conc. NA 20 pg/l 0.6 M 0.6 M NA

Description
The aim of Alleles iPS RT-PCR primer sets is to provide researches with an efficient tools to initiate work in the iPS or ES field. 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 defined and tested in Takahashi et al. 2007 [1], are conveniently and sufficiently provided for 50 reactions for each gene. Each batch of primers is vigorously tested for oligo integrity.

Template DNA (1ng/l)

M)

Upstream primer (20

Downstream genespecific primer (20 M) Nuclease-free water

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

Features
Pre-tested and complete set of RT-PCR primers for: 25 stem cell marker genes and 18 germ layer specific 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 demonstrated human iPS for the first time. All oligos were produced in-house at Allele Biotech with quality control provided at uniform concentrations for easy reaction setup. Additional controls are included

Box 3 | Recommended PCR Setup

Component Taq DNA Polymerase Template DNA (1ng/l)
M)

Volume 5 units 1l 1 l 1 l 5 l 1 l

Final Conc. NA 20 pg/l 0.6 M 0.6 M 1X 0.2 mM

Upstream primer (20

Downstream genespecific primer (20 M) 10 X PCR buffer 10 mM dNTP mix Nuclease-free water

Box 1 | Product Info Human iPS RT-PCR Primer Set

CAT# ABP-SC-iPShRES

add to final volume of 50 l

Contents
dT(20) primer for reverse transcription (50 l at 20 M) 45 pairs of RT-PCR primers
Primers are provided for 25 ES marker genes, 8 germ 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 amplification (2-step) as follows: Denature 94C for 30 sec Anneal 58C for 30 sec Anneal and extend 72C for 1min

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1.

Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblast by defined factors. Cell, 2007. 131(5): p. 861-72.

References

or 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 Biotech is strictly prohibited

Table 1 | Human iPS RT-PCR Primers

Symbol Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell OCT3/4 (POU5F1) SOX2 NANOG GDF3 ZFP42 (REX1) FGF4 ESG1 (DPPA5) DPPA4 DPPA2 TERT KLF4 c-Myc DNMT3B GABRB3 TDGF1 GAL LEFTY1 (LEFTB) IFITM1 NODAL UTF1 LEFTY2 (EBAF) GRB7 Accession NM_002701.4 Primers Sequence hOCT3/4-S1165:GACAGGGGGAGGGGAGGAGCTAGG hOCT3/4-AS1283:CTTCCCTCCAACCAGTTGCCCCAAAC NM_003106.2 NM_024865.2 NM_020634.1 NM_174900.3 NM_002007.2 NM_00102529 0.1 NM_018189.3 NM_138815.2 NM_198253.2 NM_004235.4 NM_002467.3 NM_006892.3 NM_021912.3 NM_003212.2 NM_015973.3 NM_020997.2 NM_003641.3 NM_018055.4 NM_003577.2 NM_003240.2 NM_005310.2 hSOX2-S1430:GGGAAATGGGAGGGGTGCAAAAGAGG hSOX2-AS1555:TTGCGTGAGTGTGGATGGGATTGGTG ECAT4-macaca-968S:CAGCCCCGATTCTTCCACCAGTCCC ECAT4-macaca-1334AS:CGGAAGATTCCCAGTCGGGTTCACC hGDF3-S243:CTTATGCTACGTAAAGGAGCTGGG hGDF3-AS850:GTGCCAACCCAGGTCCCGGAAGTT hREX1-RT-U:CAGATCCTAAACAGCTCGCAGAAT hREX1-RT-L:GCGTACGCAAATTAAAGTCCAGA hFGF4-RT-U:CTACAACGCCTACGAGTCCTACA hFGF4-RT-L:GTTGCACCAGAAAAGTCAGAGTTG hpH34-S40:ATATCCCGCCGTGGGTGAAAGTTC hpH34-AS259:ACTCAGCCATGGACTGGAGCATCC hECAT15-1-S532:GGAGCCGCCTGCCCTGGAAAATTC hECAT15-1-AS916:TTTTTCCTGATATTCTATTCCCAT hECAT15-2-S85:CCGTCCCCGCAATCTCCTTCCATC hECAT15-2-AS667:ATGATGCCAACATGGCTCCCGGTG hTERT-S3234:CCTGCTCAAGCTGACTCGACACCGTG hTERT-AS3713:GGAAAAGCTGGCCCTGGGGTGGAGC hKLF4-AS1826:TGATTGTAGTGCTTTCTGGCTGGGCTCC hKLF4-S1128:ACGATCGTGGCCCCGGAAAAGGACC hMYC-S253:GCGTCCTGGGAAGGGAGATCCGGAGC hMYC-AS555:TTGAGGGGCATCGTCGCGGGAGGCTG hDNMT3B-S2502:TGCTGCTCACAGGGCCCGATACTTC hDNMT3B-S2716:TCCTTTCGAGCTCAGTGCACCACAAAAC hGABRB3-S1029:CCTTGCCCAAAATCCCCTATGTCAAAGC hGABRB3-AS1280:GTATCGCCAATGCCGCCTGAGACCTC hTDGF1-S490:CTGCTGCCTGAATGGGGGAACCTGC hTDGF1-AS700:GCCACGAGGTGCTCATCCATCACAAGG hGAL-S415:TGCGGCCCGAAGATGACATGAAACC hGAL-AS579:CCCAGGAGGCTCTCAGGACCGCTC hLEFTB-S794:CTTGGGGACTATGGAGCTCAGGGCGAC hLEFTB-AS1023:CATGGGCAGCGAGTCAGTCTCCGAGG hIFITM1-S166:CCCCAAAGCCAGAAGATGCACAAGGAG hIFITM1-AS368:CGTCGCCAACCATCTTCCTGTCCCTAG hNODAL-S693:GGGCAAGAGGCACCGTCGACATCA hNODAL-AS900:GGGACTCGGTGGGGCTGGTAACGTTTC hUTF1-S832:CCGTCGCTGAACACCGCCCTGCTG hUTF1-AS979:CGCGCTGCCCAGAATGAAGCCCAC hEBAF-S782:GCTGGAGCTGCACACCCTGGACCTCAG hEBAF-AS1032:GGGCAGCGAGGCAGTCTCCGAGGC hGRB7-S1250:CGCCTCTTCAAGTACGGGGTGCAGCTGT hGRB7-AS1467:TGGGCAGGCTGAGGCGGTGGTTTG 241 bp 274 bp 171 bp 234 bp 229 bp 255 bp 188 bp 237 bp 277 bp 242 bp 328 bp 397 bp 446 bp 608 bp 408 bp 243 bp 371 bp 306 bp 631 bp 391 bp 151 bp Size 144 bp
Applications

Endo OCT3/4 RT-PCR Endo SOX2 RT-PCR NANOG RT-PCR GDF3 RT-PCR REX1 RT-PCR FGF4 RT-PCR ESG1/DPPA5 RT-PCR DPPA4 RT-PCR DPPA2 RT-PCR hTERT RT-PCR Endo KLF4 RT-PCR Endo c-MYC RT-PCR DNMT3B RT-PCR GABRB3 RT-PCR TDGF1 RT-PCR GAL RT-PCR LEFTB RT-PCR IFITM1 RT-PCR NODAL RT-PCR UTF1 RT-PCR EBAF RT-PCR

GRB7 RT-PCR

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Table 2 | Human iPS RT-PCR Primers

Undifferentiated ES cell Undifferentiated ES cell Undifferentiated ES cell Mesoderm MSX1 NM_002448.3 BXDC2 NM_018321.3 CD9 NM_001769.2 PODXL NM_005397.3 hPODXL-S1204:TCCAGCCCCACAGCAGCATCAACTACC hPODXL-AS1403:CCGGGTTGAAGGTGGCTTTGACTGCTC hCD9-S369:GTGCATGCTGGGACTGTTCTTCGGCTTC hCD9-AS564:CACGCCCCCAGCCAAACCACAGCAG hBRIX-S596:CACCACGGTATCATCCCAAAAGCCAACC hBRIXAS798:ACGCCGATGCATGTTTGGTGACTGGTAG hMSX1-S665:CGAGAGGACCCCGTGGATGCAGAG hMSX1-AS938:GGCGGCCATCTTCAGCTTCTCCAG Mesoderm T, brachyury homolog (mouse) Ectoderm PAX6 NM_001604.4 hPAX6-S1206:ACCCATTATCCAGATGTGTTTGCCCGAG hPAX6-AS1497:ATGGTGAAGCTGGGCATAGGCGGCAG Endoderm FOXA2 NM_153675.2 hFOXA2-S208:TGGGAGCGGTGAAGATGGAAGGGCAC hFOXA2-AS398:TCATGCCAGCGCCCACGTACGACGAC Endoderm SOX17 NM_022454.3 hSOX17-S423:CGCTTTCATGGTGTGGGCTAAGGACG hSOX17-AS583:TAGTTGGGGTGGTCCTGCATGTGCTG Endoderm AFP NM_001134.1 hAFP-S948:GAATGCTGCAAACTGACCACGCTGGAAC hAFP-AS1201:TGGCATTCAAGAGGGTTTTCAGTCTGGA Endoderm KRT8(CK8) NM_002273.3 hCK8-S734:CCTGGAAGGGCTGACCGACGAGATCAA hCK8-AS9 56:CTTCCCAGCCAGGCTCTGCAGCTCC Endoderm KRT18 (CK18) Ectoderm Neuron Marker Dopaminergic Neuron Markers Dopaminergic Neuron Markers Dopaminergic Neuron Markers Dopaminergic Neuron Markers Dopaminergic Neuron Markers Cardiomyocyte Markers Cardiomyocyte Markers Cardiomyocyte Markers Cardiomyocyte Markers Myosi (MYHCB) NM_000257.2 MYL7 (MYL2A) TNNT2 (TnTc) MEF2C NM_00100143 2.1 NM_002397.3 NM_021223.2 SLC6A3/ DAT LMX1B NM_002316.2 NM_001044.3 DDC (AADC) CHAT NM_00108297 1.1 NM_020984.3 GFAP MAP2 NM_00103953 8.1 NM_002055.3 NM_199187.1 hCK18-S1125:AGCTCAACGGGATCCTGCTGCACCTTG hCK18AS1322:CACTATCCGGCGGGTGGTGGTCTTTTG hMAP2-S5401:CAGGTGGCGGACGTGTGAAAATTGAGAGTG hMAP2-AS5587:CACGCTGGATCTGCCTGGGGACTGTG hGFAP-S1040:GGCCCGCCACTTGCAGGAGTACCAGG hGFAPAS1342:CTTCTGCTCGGGCCCCTCATGAGACG hAADC-S1378:CGCCAGGATCCCCGCTTTGAAATCTG hAADCAS1594:TCGGCCGCCAGCTCTTTGATGTGTTC hChAT-S1360:GGAGGCGTGGAGCTCAGCGACACC hChAT-AS1 592:CGGGGAGCTCGCTGACGGAGTCTG hDAT-S1935:ACAGAGGGGAGGTGCGCCAGTTCACG hDAT-AS2 207:ACGGGGTGGACCTCGCTGCACAGATC hLMX1B-S770:GGCACCAGCAGCAGCAGGAGCAGCAG hLMX1B-AS1020:CCACGTCTGAGGAGCCGAGGAAGCAG hMYL2A-S258GGGCCCCATCAACTTCACCGTCTTCC hMYL2AAS468TGTAGTCGATGTTCCCCGCCAGGTCC hTnTc-S524:ATGAGCGGGAGAAGGAGCGGCAGAAC hTnTcAS730:TCAATGGCCAGCACCTTCCTCCTCTC hMEF2C-S1407:TTTAACACCGCCAGCGCTCTTCACCTTG hMEF2C-AS1618:TCGTGGCGCGTGTGTTGTGGGTATCTCG hMYHCB-S5582:CTGGAGGCCGAGCAGAAGCGCAACG hMYHCB-AS5815:GTCCGCCCGCTCCTCTGCCTCATCC 258 bp 239 bp MEF2C RTPCR MYHCB RTPCR 238 bp 236 bp 276 bp 298 bp SLC6A3/DAT RT-PCR LMX1B RTPCR MYL2A RTPCR TnTc RT-PCR 256 bp ChAT RT-PCR 242 bp AADC RT-PCR 328 bp 212 bp MAP2 RT-PCR GFAP RT-PCR 233 bp CK18 RT-PCR 247 bp 281 bp 608 bp 216 bp 317 bp PAX6 RT-PCR FOXA2 RT-PCR SOX17 RT-PCR AFP RT-PCR CK8 RT-PCR NM_003181.2 hBRACHYURY-S1292:GCCCTCTCCCTCCCCTCCACGCACAG hBRACHYURY-AS1540:CGGCGCCGTTGCTCACAGACCACAGG 274 bp 307 bp MSX1 RT-PCR BRACHYURY/T RT-PCR 230 bp 220 bp 226 bp

PODXL RT-PCR
CD9 RT-PCR BRIX 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.
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