iPS RT-PCR Primer Set - Allele...
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CAT# ABP-SC-iPSmRES Storage conditions: -20 °C Background Stem 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 Description The 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 26 marker genes, with housekeeping control. - All sequences are identical to those as published by Takahashi et al. in their 2006’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. Applications Suitable for analyzing the reprogramming or differentiation stage of iPS or ES cells. Quality Assurance Each 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) -- 29 pairs of RT-PCR primers Primers are provided for 21 ES marker genes, 2 derm layer markers, 3 other markers, and 3 control house-keeping genes, as in [1]. Information is provided in Table 1 on the next page. Recommended Protocols RT-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 Concentration Template DNA (1ng/µl) 1µl 20 pg/µl Upstream primer (20 µM) 1 µl 0.6 µM Downstream gene- 1 µl 0.6 µM Distilled water 21 µl Allele-in-one Mix 25 µl The following procedure is suggested as a starting point when using Taq polymerase: Component Volume Final Concentration Template DNA (1ng/µl) 1 µl 20 pg/µl 10 X PCR buffer 5 µl 1X 10 mM dNTP mix 1 µl 0.2 mM Upstream primer (20 µM) 1 µl 0.6 µM Downstream gene- 1 µl 0.6 µM Taq DNA Polymerase 5 unit Distilled water follows: Denature 94°C for 30 sec Anneal 58°C for 30 sec Anneal and extend 72°C for 1min 1. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells Cell, 2006. 126(4): p. 663-76. 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-PCR iPS RT-PCR Primer Set Primer 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: [email protected]
Transcript of iPS RT-PCR Primer Set - Allele...
CAT# ABP-SC-iPSmRESStorage 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 26 marker genes, with housekeeping control.
- All sequences are identical to those as published by Takahashi et al. in their 2006’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)
-- 29 pairs of RT-PCR primers
Primers are provided for 21 ES marker genes, 2 derm layer markers, 3 other markers, and 3 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. and S. Yamanaka, Induction of pluripotent stem cells
Cell, 2006. 126(4): p. 663-76.
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: [email protected]
Symbol Accession Primers Size Applications
ES cell
marker
Ecat1 NM_025890.3 Ecat1-RT-S: TGTGGGGCCCTGAAAGGCGAGCTGAGAT
Ecat1-RT-AS: ATGGGCCGCCATACGACGACGCTCAACT
164 bp RT-PCR for Ecat1
ES cell
marker
Esg1
(Dppa5a)
NM_025274.3 pH34-U38: GAAGTCTGGTTCCTTGGCAGGATG
pH34-L394: ACTCGATACACTGGCCTAGC
376 bp RT-PCR for Esg1
ES cell
marker
Rex1
(Zfp42)
NM_009556.3 Rex1-RT-S: ACGAGTGGCAGTTTCTTCTTGGGA
Rex1-RT-AS: TATGACTCACTTCCAGGGGGCACT
287 bp RT-PCR for Rex1
ES cell
marker
Utf1 NM_009482.2 Utf1-RT-S: GGATGTCCCGGTGACTACGTCTG
Utf1-RT-AS: GGCGGATCTGGTTATCGAAGGGT
344 bp RT-PCR for Utf1
ES cell
marker
Cripto NM_011562.2 Cripto-S: ATGGACGCAACTGTGAACATGATGTTCGCA
Cripto-AS: CTTTGAGGTCCTGGTCCATCACGTGACCAT
174 bp RT-PCR for Cripto
ES cell
marker
Dax1 NM_007430.4 Dax1-S1096: TGCTGCGGTCCAGGCCATCAAGAG
Dax1-AS1305: GGGCACTGTTCAGTTCAGCGGATC
233 bp RT-PCR for Dax1
ES cell
marker
Zfp296 NM_022409.1 Zfp296-S67: CCATTAGGGGCCATCATCGCTTTC
Zfp296-AS350: CACTGCTCACTGGAGGGGGCTTGC
307 bp RT-PCR for Zfp296
ES cell
marker
Nat1
(Eif4g2）
NM_013507.3 Nat1-U283: ATTCTTCGTTGTCAAGCCGCCAAAGTGGAG
Nat1-L476: AGTTGTTTGCTGCGGAGTTGTCATCTCGTC
223 bp RT-PCR for Nat1
ES cell
marker
c-Myc NM_010849.4 c-Myc-S1093: CAGAGGAGGAACGAGCTGAAGCGC
m-cMyc-AS: TTATGCACCAGAGTTTCGAAGCTGTTCG
228 bp RT-PCR for
Total c-Myc
ES cell
marker
c-Myc NM_010849.4 Myc-S1904: TGACCTAACTCGAGGAGGAGCTGGAATC
Myc-AS2042: AAGTTTGAGGCAGTTAAAATTATGGCTGAAGC
170 bp RT-PCR for
Endogenous c-Myc
ES cell
marker
Nanog NM_028016.2 6047-S4: AGGGTCTGCTACTGAGATGCTCTG
6047-AS: 5CAACCACTGGTTTTTCTGCCACCG
228 bp RT-PCR for
Total Nanog
ES cell
marker
Nanog NM_028016.1* 6047-S1: CAGGTGTTTGAGGGTAGCTC
6047-AS1: CGGTTCATCATGGTACAGTC
223 bp RT-PCR for
Endogenous Nanog
ES cell
marker
Eras NM_181548.2 45328-S118: ACTGCCCCTCATCAGACTGCTACT
ERas-AS304: CACTGCCTTGTACTCGGGTAGCTG
210 bp RT-PCR for ERas
ES cell
marker
Fgf4 NM_010202.5 Fgf4-RT-S: CGTGGTGAGCATCTTCGGAGTGG
Fgf4-RT-AS: CCTTCTTGGTCCGCCCGTTCTTA
197 bp RT-PCR for Fgf4
ES cell
marker
Oct3/4 NM_013633.2 Oct3/4-S9: TCTTTCCACCAGGCCCCCGGCTC
Oct3/4-AS210: TGCGGGCGGACATGGGGAGATCC
224 bp RT-PCR for
Endogenous Oct3/4
ES cell
marker
Oct3/4 NM_013633.2 Oct3/4-U474: CTGAGGGCCAGGCAGGAGCACGAG
Oct3/4-L935: CTGTAGGGAGGGCTTCGGGCACTT
485 bp RT-PCR for
Total Oct3/4
ES cell
marker
Gdf3 NM_008108.4 Gdf3-U253: GTTCCAACCTGTGCCTCGCGTCTT
Gdf3-L16914: AGCGAGGCATGGAGAGAGCGGAGCAG
570 bp RT-PCR for Gdf3
ES cell
marker
Sox2 NM_011443.3 Sox2-S768: GGTTACCTCTTCCTCCCACTCCAG
anti-Sox2-AS: TCACATGTGCGACAGGGGCAG
193 bp RT-PCR for
Total Sox2
ES cell
marker
Sox2 NM_011443.3 Sox2-RT-S: TAGAGCTAGACTCCGGGCGATGA
Sox2-RT-AS: TTGCCTTAAACAAGACCACGAAA
297 bp RT-PCR for
Endogenous Sox2
ES cell
marker
Klf4 NM_010637.3 mKLF4cDNAs1064: CACCATGGACCCGGGCGTGGCTGCCAGAAA
mKLF4cDNAas1769: TTAGGCTGTTCTTTTCCGGGGCCACGA
739 bp RT-PCR for
Total Klf4
ES cell
marker
Klf4 NM_010637.3 Klf4-S1236: GCGAACTCACACAGGCGAGAAACC
Klf4cDNAas2547: TCGCTTCCTCTTCCTCCGACACA
711 bp RT-PCR for
Endogenous Klf4
Gata6 NM_010258.3 Gata6-S917: ACCTTATGGCGTAGAAATGCTGAGGGTG
Gata6-AS1250: CTGAATACTTGAGGTCACTGTTCTCGGG
334 bp RT-PCR for Gata6
TABLE 1: Mouse iPS RT-PCR PrimersTABLE 1: Mouse iPS RT-PCR Primers
Mesoderm Brachyury NM_009309.2 T-S764: ATGCCAAAGAAAGAAACGAC
T-AS1579: AGAGGCTGTAGAACATGATT
835 bp RT-PCR for Brachyury
Cdx2 NM_007673.3 Cdx2-S775: GGCGAAACCTGTGCGAGTGGATGCGGAA
Cdx2-AS1210: GATTGCTGTGCCGCCGCCGCTTCAGACC
493 bp RT-PCR for Cdx2
Ectoderm Map2 ** Mtap2-S629: CATCGCCAGCCTCGGAACAAACAG
Mtap2-AS867: TGC GCA AAT GGA ACT GGA GGC AAC
bp RT-PCR for Map2
Neo Neo-S63: GCTATTCGGCTATGACTGGGCACA
Neo-AS: 581CCACCATGATATTCGGCAAGCAGG
Bp RT-PCR for Neo
*This record has been replaced by NM_028016.2
** This gene does not seem to match with anything in the GenBank.
Other bases in red show discrepancies with GenBank files.
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