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

primers for 26 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. 2006 [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: 26 marker genes with housekeeping control. All sequences are identical to those as published by Takahashi et al. in their 2006 publication in Cell that demonstrated mouse 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

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

CAT# ABP-SC-iPSmRES

10 mM dNTP mix Nuclease-free water

add to final volume of 50 l

Contents
dT(20) primer for reverse transcription (50 l at 20 M) 29 pairs of RT-PCR primers
Primers are provided for 21 ES marker genes, 2 germ layer markers, 3 other markers, and 3 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. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.

References

Table 1 | Mouse iPS RT-PCR Primers

Symbol ES cell Marker ES cell Marker ES cell Marker ES cell Marker ES cell Marker ES cell Marker ES cell Marker ES cell Marker ES cell Marker ES cell Marker Ecat1 Esg1 (Dppa5a) Rex1 (Zfp42) Utf1 Cripto Dax1 Zfp296 Nat1 (Eif4g2) c-Myc c-Myc Accession NM_025890.3 NM_025274.3 NM_009556.3 NM_009482.2 NM_011562.2 NM_007430.4 NM_022409.1 NM_013507.3 NM_010849.4 NM_010849.4

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
Primers Sequence Size 164 bp 376 bp 287 bp 344 bp 174 bp 233 bp 307 bp 223 bp 228 bp 170 bp
Applications

Ecat1-RT-S: TGTGGGGCCCTGAAAGGCGAGCTGAGAT Ecat1-RT-AS: ATGGGCCGCCATACGACGACGCTCAACT pH34-U38: GAAGTCTGGTTCCTTGGCAGGATG pH34-L394: ACTCGATACACTGGCCTAGC Rex1-RT-S: ACGAGTGGCAGTTTCTTCTTGGGA Rex1-RT-AS: TATGACTCACTTCCAGGGGGCACT Utf1-RT-S: GGATGTCCCGGTGACTACGTCTG Utf1-RT-AS: GGCGGATCTGGTTATCGAAGGGT Cripto-S: ATGGACGCAACTGTGAACATGATGTTCGCA Cripto-AS: CTTTGAGGTCCTGGTCCATCACGTGACCAT Dax1-S1096: TGCTGCGGTCCAGGCCATCAAGAG Dax1-AS1305: GGGCACTGTTCAGTTCAGCGGATC Zfp296-S67: CCATTAGGGGCCATCATCGCTTTC Zfp296-AS350: CACTGCTCACTGGAGGGGGCTTGC Nat1-U283: ATTCTTCGTTGTCAAGCCGCCAAAGTGGAG Nat1-L476: AGTTGTTTGCTGCGGAGTTGTCATCTCGTC c-Myc-S1093: CAGAGGAGGAACGAGCTGAAGCGC m-cMyc-AS: TTATGCACCAGAGTTTCGAAGCTGTTCG Myc-S1904: TGACCTAACTCGAGGAGGAGCTGGAATC Myc-AS2042: AAGTTTGAGGCAGTTAAAATTATGGCTGAAGC

RT-PCR for Ecat1 RT-PCR for Esg1 RT-PCR for Rex1 RT-PCR for Utf1 RT-PCR for Cripto RT-PCR for Dax1 RT-PCR for Zfp296 RT-PCR for Nat1 RT-PCR for Total c-Myc RT-PCR for Endogenous c-Myc RT-PCR for

ES cell Marker ES cell Marker

Nanog Nanog

NM_028016.2 NM_028016.1*

6047-S4: AGGGTCTGCTACTGAGATGCTCTG 6047-AS: 5CAACCACTGGTTTTTCTGCCACCG 6047-S1: CAGGTGTTTGAGGGTAGCTC 6047-AS1: CGGTTCATCATGGTACAGTC

228 bp 223 bp

Total Nanog RT-PCR for Endogenous Nanog RT-PCR for ERas RT-PCR for Fgf4 RT-PCR for Endogenous Oct3/4 RT-PCR for Total Oct3/4 RT-PCR for Gdf3 RT-PCR for Total Sox2 RT-PCR for Endogenous Sox2 RT-PCR for Total Klf4 RT-PCR for Endogenous Klf4 RT-PCR for Gata6

ES cell Marker ES cell Marker ES cell Marker

Eras Fgf4 Oct3/4

NM_181548.2 NM_010202.5 NM_013633.2

45328-S118: ACTGCCCCTCATCAGACTGCTACT ERas-AS304: CACTGCCTTGTACTCGGGTAGCTG Fgf4-RT-S: CGTGGTGAGCATCTTCGGAGTGG Fgf4-RT-AS: CCTTCTTGGTCCGCCCGTTCTTA Oct3/4-S9: TCTTTCCACCAGGCCCCCGGCTC Oct3/4-AS210: TGCGGGCGGACATGGGGAGATCC

210 bp 197 bp 224 bp

ES cell Marker ES cell Marker ES cell Marker ES cell Marker

Oct3/4 Gdf3 Sox2 Sox2

NM_013633.2 NM_008108.4 NM_011443.3 NM_011443.3

Oct3/4-U474: CTGAGGGCCAGGCAGGAGCACGAG Oct3/4-L935: CTGTAGGGAGGGCTTCGGGCACTT Gdf3-U253: GTTCCAACCTGTGCCTCGCGTCTT Gdf3-L16914: AGCGAGGCATGGAGAGAGCGGAGCAG Sox2-S768: GGTTACCTCTTCCTCCCACTCCAG anti-Sox2-AS: TCACATGTGCGACAGGGGCAG Sox2-RT-S: TAGAGCTAGACTCCGGGCGATGA Sox2-RT-AS: TTGCCTTAAACAAGACCACGAAA

485 bp 570 bp 193 bp 297 bp

ES cell Marker

Klf4

NM_010637.3

mKLF4cDNAs1064: CACCATGGACCCGGGCGTGGCTGCCAGAAA mKLF4cDNAas1769: TTAGGCTGTTCTTTTCCGGGGCCACGA

739 bp

ES cell Marker

Klf4

NM_010637.3

Klf4-S1236: GCGAACTCACACAGGCGAGAAACC Klf4cDNAas2547: TCGCTTCCTCTTCCTCCGACACA

711 bp

Gata6

NM_010258.3

Gata6-S917: ACCTTATGGCGTAGAAATGCTGAGGGTG Gata6-AS1250: CTGAATACTTGAGGTCACTGTTCTCGGG

334 bp

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Table 2 | Mouse iPS RT-PCR Primers

Mesoderm Brachyury Cdx2 NM_009309.2 NM_007673.3 T-S764: ATGCCAAAGAAAGAAACGAC T-AS1579: AGAGGCTGTAGAACATGATT Cdx2-S775: GGCGAAACCTGTGCGAGTGGATGCGGAA Cdx2-AS1210: GATTGCTGTGCCGCCGCCGCTTCAGACC Ectoderm Map2 493 bp 835 bp RT-PCR for Brachyury RT-PCR for Cdx2

**

Mtap2-S629: CATCGCCAGCCTCGGAACAAACAG Mtap2-AS867: TGC GCA AAT GGA ACT GGA GGC AAC

` `

RT-PCR for Map2 RT-PCR for Neo

Neo

Neo-S63: Neo-AS: GCTATTCGGCTATGACTGGGCACA

Neo-AS: 581CCACCATGATATTCGGCAAGCAGG

*This record has been replaced by NM_028016.2 ** This gene does not match with anything in GenBank.

Note: Other bases in red show discrepancies with GenBank files.

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